Preparation of a Fused-Silica Capillary Column

Most users of a modern capillary column regard it as a high-precision and sophis- ticated device and purchase columns from a vendor. Few give any thought to the steps involved in column preparation. Their number one priority is understand- ably the end result of accurate and reproducible chromatographic data that the column can provide. In this section, an overview of deactivation and coating of a fused-silica column with stationary phase is discussed.

3.9.2.1 Silanol Deactivation

For maximum column performance, blank or raw fused-silica tubing must receive pretreatment prior to the final coating with stationary phase. The purpose of pretreatment is twofold: to cover up or deactivate active surface sites and to create a surface more wettable by the phase. The details of the procedure are dependent on the stationary phase to be subsequently coated, but deactivation

(a)

FIGURE 3.20 Photographs of metal-clad capillary columns: (a) aluminum-clad cap- illary column (photograph courtesy of the Quadrex Corp.); (b) fused-silica-lined stain- less-steel capillary column (lower) and polyimide-clad fused-silica capillary columns (upper); photographs courtesy of the Restek Corporation.

(b)

FIGURE 3.20 (Continued )

is essential for producing a column having a uniform film deposition along the inner wall of the capillary.

Although metal ions are not a factor with fused silica, the presence of silanol groups still must be addressed; otherwise the column has residual surface activity.

Column activity can be demonstrated in several ways. The chromatographic peak of a given solute can completely disappear, partially disappear as its size dimin- ishes, or exhibit tailing. A chromatogram of a test mixture showing the activity of an uncoated fused-silica column is displayed in Figure 3.21a; the inherent acidity associated with surface silanol groups is responsible for the complete dis- appearance of the basic probe solute, 2,6-dimethylaniline. When this column is deactivated with a precoating of Carbowax 20M, the residual surface column is considerably reduced (Figure 3.21b).

(a) (b)

0 2 4 6 8

Time (minutes)

0 2 4 6 8

Time (minutes) 1

2

3 4

5

6 7

8 9

FIGURE 3.21 Chromatograms of an activity mixture on 15-m×0.25-mm (a) uncoated fused silica and (b) fused-silica capillary column after deactivation with Carbowax 20M.

Column temperature: 70^◦C; 25 cm/s He; split injection (100 – 1). Peaks: (1)n-dodecane, (2)n-tridecane, (3) 5-nonanone, (4)n-tetradecane, (5)n-pentadecane, (6) 1-octanol, (7) naphthalene, (8) 2,6-dimethylaniline, and (9) 2,6-dimethylphenol. (Reference 7.)

A variety of agents and procedures have been explored for deactivation purposes (60–74). For subsequent coating with nonpolar and moderately polar stationary phases such as polysiloxanes, fused silica has been deactivated by silylation at elevated temperatures, thermal degradation of polysiloxanes and polyethylene glycols, and the dehydrocondensation of silicon hydride polysiloxanes (71,75–79).

Blomberg has published a comprehensive review of deactivating methods using polysiloxanes (80). One approach has been suggested by Schomburg et al.

(77), who prepared columns having excellent thermal stability with polysilox- ane liquid phases as deactivators and proposed that the decomposition prod- ucts formed at the elevated temperatures chemically bond to surface silanols.

Surface stationary-phase compatibility has also been achieved with cyclic silox- anes having the same side functional groups as the silicone stationary phase.

Octamethylcyclotetrasiloxane (D₄) has been decomposed at 400^◦C by Stark et al.

(81), who postulated that the process involved opening the D₄ring to form a 1,4- hydroxyoctamethyltetrasiloxane. They indicate that a terminal hydroxyl group interacts with a protruding silanol group eliminating water, and in a secondary reaction the other hydroxyl reacts with another silanol or even a tetrasiloxane.

Well-deactivated capillary columns can be prepared by this technique (82). In Figure 3.22 the effectiveness of the D₄ deactivation procedure is demonstrated for both acidic and basic test mixtures where the components have excellent band profiles. Woolley et al. outlined an easily implemented deactivation proce- dure employing the thermal degradation of polyhydrosiloxane at about 260^◦C, where the silyl hydride groups undergo reaction with surface silanols to form rather stable Si–O–Si bonds and also hydrogen gas (78). This method has the merits of a reaction time less than a hour, a relatively low reaction temperature, and a high degree of reproducibility. A representation of selected deactivated surface textures is displayed in Figure 3.23.

Carbowax 20M has also been successfully used to deactivate column sur- faces (84,85). After coating a thin film of Carbowax 20M, for example, on the column wall, the column is heated to 280^◦C, then exhaustively extracted with

C₈ –OH

C₁₀ –NH₂

C₁₂ –NH₂ C₈ –NH₂

C₈–loHl₂

C8–OOHl2

C₁₀ C₁₀

C₁₂

C₁₈ C₁₂

C₁₈ Cl

Cl Cl

OH OH

NO₂

Temp(°c) 40

Time(min.) 0 10 20 30

100 150 Temp(°c) 40

Time(min.) 0 10 20 30

100 150

(a) (b)

FIGURE 3.22 Chromatogram illustrating the inertness attainable on a D₄-deactivated SE-54 (0.25-µm) fused-silica column with (a) an acidic test mixture and (b) a basic test mixture; temperature programmed from 40^◦C at 4^◦C/min after 2 min isothermal hold; H₂ carrier gas at 45 cm/s (reproduced from Reference 82 and reprinted with permission of Elsevier Science Publishers).

FIGURE 3.23 Selected reagents used for deactivation of silanol groups: (a) disilazanes, (b) cyclic siloxanes, (c) silicon hydride polysiloxanes. Lower portion is a view of fused- silica surface with (d) adsorbed water (e) after deactivation with a trimethylsilylating reagent and (f) after treatment with a silicon hydride polysiloxane. (Reproduced from Reference 83 and reprinted with permission from Elsevier Science Publishers.)

solvent, leaving a nonextractable film of Carbowax 20M on the surface. Both apolar and polar stationary phases, including Carbowax, can then be coated on capillaries subjected to this pretreatment (86). Dandeneau and Zerenner used this procedure to deactivate their first fused-silica columns (45). Other polyethylene glycols used for deactivating purposes have been Carbowax 400 (87), Carbowax 1000 (88), and Superox-4 (89). Moreover, when a polar polymer is used for deactivation, it may alter the polarity of the stationary phase, and this effect becomes particularly problematic with a thin film of a nonpolar phase where the resulting phase has retentions of a mixed phase. Furthermore, silazanes and cyclic silazanes, as deactivating agents, ultimately yield a basic final col- umn texture, whereas chlorosilanes, alkoxysilanes hydrosilanes, hydrosiloxanes, siloxanes, and Carbowax produce an acidic column (4). In essence, a deacti- vation procedure imparts different residual surface characteristics and is often selected with the stationary phase as well as the application in mind. Many col- umn manufacturers offer base-deactivated columns (and base-deactivated inlet liners) with several stationary phases for successful chromatography of amines.

There have been two additional approaches to deactivation: (1) the coating of a layer of polypyrrone on the inner surface of the tubing prior to the deposi- tion of the stationary phase, thereby circumventing the temperature limitation

of polyimide in high temperature applications (90); and (2) the “Siltek” process, where deactivation is achieved via a vapor deposition process (91,92) as opposed to procedures using agents such as liquid silazane or chlorosilane. An alternative procedure is the utilization of OH-terminated stationary phases where deactivation and immobilization of the phase occurs in a single-step process (Section 3.11.5).

3.9.2.2 Static Coating of Capillary Columns

The goal in coating a capillary column is the uniform deposition of a thin film, ranging from 0.1 to 8µm in thickness, on the inner wall of a length of clean, deactivated fused-silica tubing. Jennings (94) has reviewed the various methods for coating stationary phases. The static method of coating is discussed here because it is most widely used today by column manufacturers.

This procedure was first described by Bouche and Verzele (95), who initially completely filled the column with a solution of known concentration of stationary phase. In this procedure, one end of the column is sealed and the other is attached to a vacuum source. As the solvent evaporates, a uniform film is deposited on the column wall. The column must be maintained at constant temperature for uniform film deposition. The coating solution should be free of microparticu- lates and dust, be degassed so no bumping occurs during solvent evaporation, and there should be no bubbles in the column. Pentane is the recommended sol- vent because of its high volatility and should be used wherever stationary phase solubility permits. Evaporation time is approximately half that required to evap- orate methylene chloride. The static coating technique offers the advantage of an accurate determination of the phase ratio (Section 3.10.3) from which the film thickness of the stationary phase can be calculated.

3.9.2.3 Capillary Cages

Since the ends of flexible fused-silica capillary tubing are inherently straight, columns must be coiled and confined on a circular frame, also called a “cage”

(Figure 3.20). The capillary column can then be mounted securely in the column oven of a gas chromatograph. Fused-silica capillary columns of 0.10–0.32 mm i.d. are wound around a 5- or 7-in.-diameter cage whereas an 8-in. cage is used with megabore columns (0.53 mm i.d.). Installation of a capillary column is greatly facilitated, since the ends of a fused-silica column can easily be inserted at the appropriate recommended lengths into sample inlets and detector systems. The ultimate in gas chromatographic system inertness is attainable with on-column injection, where a sample encounters only fused silica from the point of injection to the tip of a FID flame jet.

3.9.2.4 Test Mixtures for Monitoring Column Performance

The performance of a capillary column can be evaluated with a test mixture whose components and resulting peak shapes serve as monitors of column efficiency and diagnostic probes for adverse adsorptive effects and the acid/base character of a column. These mixtures are used by column manufactures in the quality control of their columns and are likewise recommended for the chromatographic laboratory.

A chromatogram of a test mix and a report are usually supplied with a commer- cially prepared column. Using the same indicated chromatographic conditions, the separation should be duplicated by the user prior to running samples with column. In the test report evaluating the performance of the column, chromato- graphic data are listed. These may include retention times of the components in the text mix, corresponding Kovats retention indices of several, if not all, of the solutes, the number of theoretical plates N and/or the effective plate num- ber N_eff, Trennzahl and the acid/base inertness ratio (the peak height ratio of the acidic and basic probes in the test mixture). The values of two additional chromatographic parameters, separation number (Trennzahl number) and coating efficiency, may also be included in the report; the significance of TZ has been discussed in Section 3.6.4, and coating efficiency is treated in Section 3.10.5.

The first chromatogram obtained on a new column may be viewed as the

“birth certificate” of a column and defines column performance at timet =0 in the laboratory; a test mix should also be analyzed periodically to determine any changes in column behavior occurring with age and use. For example, a column may acquire a pronouncedly basic character if it has been employed routinely for amine analyses. Another important but often overlooked aspect is that a test mixture serves to monitor the performance of the total chromatographic system, not just the performance of the column. If separations gradually deteriorate over time, the problem may not always be column-related but could be due to extra- column effects, such as a contaminated or activated inlet liner. Commonly used components, their accepted abbreviations, and functions are listed in Table 3.16.

An ideal capillary column should be well deactivated and have excellent ther- mal stability and high separation efficiency. The extent of deactivation is usually

TABLE 3.16 Test Mixture Components and Role

Probe Function

n-Alkanes, typically C10 – C15 Column efficiency; Trennzahl number (TZ) Methyl esters of fatty acids,

usually C9 – C12 (E9 – E12)

Separation number; column efficiency 1-Octanol (ol) Detection of hydrogen-bonding sites, silanol

groups

2,3-Butanediol (D) More rigorous test of silanol detection

2-Octanone Detection of activity associated with Lewis acids Nonanal(al) Aldehyde adsorption other than via hydrogen

bonding

2,6-Dimethylphenol (P) Acid – base character 2,6-Dimethylaniline (A) Acid – base character 4-Chlorophenol Acid – base character

n-Decylamine Acid – base character

2-Ethylhexanoic acid (S) More stringent measure of irreversible adsorption Dicyclohexylamine (am) More stringent measure of irreversible adsorption Note: Abbreviations of the components in the comprehensive Grob mix indicated in parentheses.

manifested by the amount of peak tailing for polar compounds. The most compre- hensive and exacting test mixture is the solution reported by Grob et al. (96) and is more sensitive to residual surface activity than other polarity mixes. Adsorp- tion may cause (1) broadened peaks of Gaussian shapes, (2) a tailing peak of more or less the correct peak area, (3) a reasonably shaped peak with reduced area, and (4) a skewed peak of correct area but having an increased retention time. Furthermore, irreversible adsorption cannot always be detected by peak shape. In the Grob procedure one measures peak heights as a percentage of that expected for complete and undistorted elution. The technique encompasses all types of peak deformations (broadening, tailing, and irreversible adsorption). A solution whose components are present at specific concentrations is analyzed under recommended column temperature programming conditions.

In practice, the percentage of the peak height is determined by drawing a line (the 100% line) connecting the peak maxima of the nonadsorbing peaks (n-alkanes and methyl esters), as shown in Figure 3.24. Alcohols are more sensi- tive than the other probes to adsorption caused by hydrogen bonding to exposed silanols. The acid and base properties are ascertained with probe solutes such as 2,6-dimethylaniline and 2,6-dimethylphenol, respectively. However, most col- umn manufacturers recommend a modification of the Grob scheme to circumvent the lengthy time involved and, instead, tailor the composition of the mix and col- umn temperature conditions to be commensurate with the particular deactivation procedure and stationary phase under consideration. A widely used test mixture consists of the components designated in Figure 3.25, where the test mix is used to also demonstrate selectivity by comparing separations on the three columns of the same dimensions but having stationary phases.

Guthrie and Harland (4) have commented that the effects of deactivation, the chemistry of the stationary phase and its crosslinking, as well as the effect of any postprocess treatment all appear in the final version of a column. An example of this situation is the separation of the Grob mixture (Figure 3.26a) performed on

10 11

al

E₁₀ am E₁₁ E₁₂ A P S

ol

0 5 10 15 20 25 30 35

TIME (minutes)

FIGURE 3.24 Chromatogram of a comprehensive Grob mixture on a 15-m× 0.32-mm-i.d. Carbowax 20M capillary column. Column conditions: 75 – 150^◦C at 1.7^◦C/min; 28 cm/s He. Designation of solutes appears in Table 3.16. (Reference 7.)

FIGURE 3.25 Chromatograms of an activity mixture on three columns of identical dimensions but different stationary phases as indicated; conditions: 15-m×0.25-mm- i.d.×0.25-µm-film capillary columns, 110^◦C, 25 cm/s He, FID.

a 15-m×0.25-mm-i.d. fused-silica capillary column deactivated with Carbowax 20M, after which the column received a recoat of the polymer. After crosslinking of the stationary phase (Figure 3.26b), column behavior changed markedly. The 2,3-butanediol peak (D), absent in Figure 3.26a, is present on the crosslinked phase that has acquired increased acidity in the crosslinking process. Note the

10 11

al

am ol E₁₀ D

A P S

E₁₁ E₁₂

10 11

al ol E₁₀am

A P S

E11 E12

(b)

(a) Time (minutes)

0 10 20 30 40 50

FIGURE 3.26 Chromatogram of a comprehensive Grob mixture on a 15-m×0.32-mm- i.d. Carbowax 20M capillary column (a) after coating and (b) after crosslinking the stationary phase. Column conditions: 75 – 150^◦C at 2^◦C/min; 28 cm/s He. Designation of solutes appears in Table 3.16. (Reference 7.)

decreased peak height of the dicyclohexylamine probe (am) and the increased peak height of 2-ethylhexanoic acid (S). Thus, any change or a minor modification in column preparation can affect the final column performance.

3.10 CHROMATOGRAPHIC PERFORMANCE