Anal. Chem. 1080, 61, 2-11
2
Synthesis and Characterization of Highly Stable Bonded Phases for High-Performance Liquid Chromatography Column Packings J. J, Kirkland,* J. L. Glajch, and R. D. Farlee Central Research and Development Department, E. I. du Pont de Nemours and Company, Experimental Station, B-228, P.O. Box 80228, Wilmington, Delaware 19880-0228
Two new classes of sllanemodffled silicas were syntheslred and characterized by chromatographic and spectroscopic techniques. These new bonded phases are signlfkantly more stable toward hydrolysis than previous bonded-phase silicas; retention and column efficiency are comparable. The first type uses bifunctional (or “bidentate”)Manes containing one reactive atom on each of two silicon atoms that connect through a bridging group such as -0- or -(CH2)”-. The second type uses a monofunctional silane with at least two bulky groups (e.g., isopropyl) on the silane silicon atom. These bulky groups provide steric protection to the SI-0-Si bond formed between the silane and the surface of the silica. The new bonded-phase silicas exhibit highly reproducible gradient elution high-performance liquid chromatography separations of peptides and protelns with low-pH mobile phases.
INTRODUCTION The development of column packings with covalent bonded phases is one of the factors that has led to the explosive growth of high-performance liquid chromatography (HPLC) during the past 15 years. Estimates of current use indicate that about three-fourths of all separations are now carried out with columns of these chemically bonded stationary phases (1). To form bonded-phase packings, silica typically is reacted with functionalized silanes according to
Si-OH
+ SiX,R4-,
-
Si-(OSiXn-lR4-n)
(1)
where X is a reactive group such as alkoxy or halide, and R commonly is an alkyl or substituted alkyl group. This reaction creates siloxane bonds between the silane and the silica atoms of the solid support, resulting in a stationary phase where the R groups of the silane largely determine the chromatographic behavior of the column packing. There are two main types of reactions possible from the general scheme of eq 1. The first type, illustrated in Figure 1,uses a monofunctional silane (n = 1)that produces a surface where the silane covers the silica as a monolayer. The advantages of this reaction are that it is reproducible and convenient; the resulting surface of monolayer coverage exhibits excellent mass-transfer properties that produce high column efficiency for most solutes. The second type of reaction uses di- or trifunctional silanes ( n = 2,3) as shown schematically in Figure 2. If the reaction is carefully controlled to exclude water, the resulting monolayer-type surface generates high column efficiency (upper portion of Figure 2). However, there are potential problems with this approach. Less than 2 equivalents of X-functionality per mole of silane can react with SiOH groups on the silica surface (2). When exposed to water (or moist air), these residual X groups on the silane hydrolyze to produce acidic SiOH groups that can be especially deleterious in the separation of basic solutes.
A silanization reaction also can produce a polymerized layer on the support surface, as shown in the lower portion of Figure 2. Polymeric bonded phases of this type have some advantages over the monolayer bonded-phase packings. First, the polymeric phases appear to be more stable to hydrolysis. We discuss this effect later in this paper. Second, polymeric phases appear to be at least partially effective in masking certain undesirable properties of the silica surface, such as residual acidic silanols. However, there are some strong disadvantages to polymeric bonded phases. These materials are more difficult to reproduce; variation in total organic coverage often is observed from one preparation to the next. Also, relatively thick stationary phases can produce poorer stationary-phase mass-transfer kinetics (3);column efficiency can be poorer than for corresponding monolayer bonded-phase packings. Finally, residual Si-OH groups in the polymer chain can be deleterious for some separations. The stability of bonded-phase packings in typical HPLC separations is often not considered to be an important problem. Many useful separations have been achieved with aqueous/organic solvent mixtures in the pH range 4-7. In this range, the covalent bonds linking the organosilane to the surface of the silica support are fairly stable, especially if the organic composition of the mobile phase is large (>20%) and the column temperature is maintained at C60 OC. These relatively mild conditions often are met in many practical applications. In these instances, bonded-phase stability is not as important as other column problems, such as plugged column frits (because of dirty samples), impurities strongly adsorbed on the column packing, etc. In recent years, reversed-phase HPLC has found wide utility for separating many basic compounds such as pharmaceuticals, agricultural chemicals, and unprotected peptides and proteins. A common approach for achieving good peak shape and retention is to separate these types of samples at lower pH (C4) with aqueous/organic phases. A typical procedure utilizes trifluoroacetic acid (TFA) in the mobile phase (4). TFA is now widely used in the reversed-phase separation of peptide and proteins because (a) samples are soluble in these solvents, (b) biomolecules produce excellent peak shapes, and (c) TFA is easily removed by lyophilization prior to further characterization of the separated species. Previous work has demonstrated that typical bonded-phase columns used for reversed-phase separations at low pH (98%). Vydac 214TP5 (Separations Group, Hesperia, CA) and U1trapore RPSC (Beckman Instruments, Berkeley, CA) columns were used as received. The Vydac columns and Ultrapore columns were 15 X 0.46 cm and 7.5 X 0.46 cm, respectively. Packings from fresh columns were characterized after unpacking an unused column from the same lot as the column used for chromatographic testing. Nucleosil columns with polymerized stationary phases were prepared by procedures detailed elsewhere (6,7). All separations were performed with LC grade methanol, acetonitrile, and water (J. T. Baker, Phillipsburg, NJ). Trifluoroacetic acid (Ionate grade, Pierce Chemical Co., Rockford, IL)was used to prepare 0.1% (by volume) solutions in both water and acetonitrile. Lysozyme, ovalbumin, insulin, glucagon, (Sigma Chemicals, St. Louis, MO), and melittin (United States Biochemicals Corp., Cleveland, OH) were test solutes; solutions of 0.5-1.0 mg/mL were prepared for the gradient elution studies.
N,N-diethylaniline, 1-phenylpentanol, 1-phenylhexane, and 1phenylheptane were from Aldrich (Milwaukee, WI); solutions of 0.05-1.7 mg/mL in 50% aqueous methanol were used with isocratic mobile phases. Procedures. The PSM-silicas were first heated in air at 540 "C and then sintered for 2 h at 850 or 975 "C (depending on starting material) to ensure high mechanical strength. The silicas were then rehydroxylated by using dilute solutions of HF in water (8). The surface area of each silica was measured by the Brunauer-Emmett-Teller (BET) method using nitrogen adsorption. Pore size distributions were determined by mercury porosimetry. The bonded-phase packings were prepared by placing a quantity of vacuum-dried silica in a flask under a dry nitrogen atmosphere. To this solid substrate was added 10 mL of dry xylene/g of silica and 2 pmol of pyridine/pmol of reactable silanol groups on the silica. The totalnumber of reactable silanols varied with the silica surface area, but 8 pmol/m2 was assumed as the silanol concentration for all rehydroxylated silicas. Ten micromoles of silane per micromole of reactable silanols was then added to the flask, and the mixture was refluxed at 138 OC for 72 h under a slow nitrogen purge. The cooled reaction mixture was filtered and the particles washed with 500 mL each of toluene, methylene chloride, methanol, 50% methanol/water, and acetone, in that order. The material was refluxed for 30 min in fresh tetrahydrofuran and fresh acetone (10 mL of each solvent/g of silica) to remove any unreacted silane. The particles then were dried
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 1, JANUARY 1, 1989
Table 11. Characterization of Column Packings"
column packing Ultrapore RPSC Vydac 2141'5 Zorbax-C-1 Nucleosil-C-1 Nucleosil-PMSC-1
k'of
surface area,
average pore,
organic
m2/g
nm
ligand
81
c-3
1.7
1.4
c-4
3.3
58
23 31d 28
2.6 0.4
86
25
86
25
78
c-1 c-1 (2-1'
carbon anal., %C initial final
1.0 1.3
9.0
0.3 1.7
1-phenylheptane initial final 3.58 5.62 15.51
7.95b 7.42c
1.06 3.55 0.80
