COLUMN PREPARATION

A

C

B

D C₁₄

C₁₆

C₁₆ C₁₈ C₁₆C₁₈

C₂₂ C₂₄

C₂₀ C₂₀ C₂₂ C₂₄

C₁₈ C₂₀ C₂₂ C₂₄ C₁₆C₁₈ C₂₀ C₂₂ C₂₄

20% at 180°c

5% at 180°c

20% at 200°c

5% at 200°c

0 10 20 30 40 50 60 0 5 10 15 20 25 30

FIGURE 3.13 Effect of concentration of stationary phase and column temperature on sample resolution (methyl esters of fatty acids). (Reproduced from Reference 20:

W. A. Supina, in Modern Practice of Gas Chromatography, 2nd ed., R. L. Grob, ed., copyright 1985, John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.)

support is too thinly coated, the exposure of active sites on the support may cause adsorption of solutes. A decrease in column temperature lowers the magnitude of the k term; however, lowering of the column temperature also decreases Dl by increasing the viscosity of the stationary phase. Effects of the various changes in chromatographic parameters on resolution that can be implemented are schematically illustrated in Figure 3.12.

A relationship between stationary phase concentration and column temperature is depicted in Figure 3.13. Decreasing column temperature increases time of analysis; in order to have the same analysis time on a heavier loaded packing in an identical column at the same flowrate requires a higher column temperature.

is employed for coating supports with high concentrations (>15%) of viscous phases, while the solution-coating method produces a more uniform phase depo- sition and is more widely utilized.

In solvent evaporation, a known amount of stationary phase is dissolved in an appropriate solvent. A weighed amount of support is added to the solution and the solvent allowed to slowly evaporate from the slurry. Since all stationary phase is deposited on the support, stirring or thorough mixing is a necessity;

otherwise nonuniform deposition of phase will result.

The technique of solution coating consists of the following steps:

1. A solution of known concentration of liquid phase in its recommended solvent is prepared.

2. A weighed amount of solid support is added to a known volume of this solution.

3. The resulting slurry is transferred into a B¨uchner funnel, with the remaining solvent removed by vacuum.

4. The volume of filtrate is measured.

5. After suction is completed, allow the wet packing to air-dry on a tray in a hood to remove residual solvent. Do not place damp packing into a laboratory drying oven!

6. The mass of liquid phase retained on the support is computed since the concentration of liquid phase in the solution is known.

This technique produces a uniform coating of a support, minimum generation of fines from the support particles, and minimum oxidation of the stationary phase.

Further details about these procedures are described in References 12 and 20.

3.7.2 Tubing Materials and Dimensions

The nature and reactivity of the sample will govern the choice of tubing for packed-column GC. Of the available materials, glass is the most inert and the best material for most applications, although somewhat fragile. Overtightening a fitting attached to a glass column can cause the dreaded “ping” sound of broken glass. Utilization of a special torque wrench, which breaks apart itself rather than the glass column when a specific torque level is exceeded, is a good investment for a gas chromatographic laboratory. Empty glass columns need deactivation or silylation prior to packing. Usually this is accomplished by filling a thoroughly cleaned empty column with a 5% solution of DMDCS in toluene. After standing for 30 min, the column is rinsed successively with toluene and methanol, then purged with dry nitrogen, after which it is ready to be packed. Moreover, as opposed to a metal column, the use of glass permits direct visualization of how well a column is packed after filling and also after the column has been used for separations. Teflon tubing, also inert, is used for the analysis of sulfur gases, halogens, HF, HCl, but has a temperature limitation of 250^◦C.

Nickel tubing offers the attractive combination of the durability and strength of metal tubing with the favorable chemical inertness of glass. Stainless steel is the next least reactive material and is utilized for analysis of hydrocarbons, permanent gases, and solvents. As is the case with all metal tubing, stainless-steel columns should be rinsed with nonpolar and polar solvents to remove residual oil and greases. When used for the analysis of polar species, a higher grade of stainless- steel tubing with a polished inner surface is recommended. Copper and aluminum tubing have been employed for noncritical separations, but their use is not rec- ommended and should be restricted to the plumbing of cylinder instrument gas lines. Oxide formation can occur on the inner surface of these materials, resulting in adsorptive tailing and/or catalytic problems under chromatographic conditions.

3.7.3 Glass Wool Plugs and Column Fittings

Chromatographic packings are retained within a column by a wad or plug of glass wool. Since the chemical nature of the wool closely resembles that of the glass column, it should also be deactivated by the same procedure used for glass columns. It is advisable to further soak the wool in a dilute solution of H3PO4

for the analysis of acidic analytes such as phenols and fatty acids. Untreated or improperly treated glass wool exhibits an active surface and can cause peak tailing. Alternatives to glass wool are stainless-steel frits and screens for gas chromatographic purposes, available from vendors of chromatographic supplies.

The ferrules and metal retaining nuts are used to form a leaktight seal of a col- umn in a gas chromatograph. Criteria for selection of the proper ferrule material are column diameter, column tubing material, maximum column temperature, and whether the connection is designated for a single use or for multiple connections and disconnections. Ferrules fabricated from various materials for metal-to-metal, glass-to-metal, and glass-to-glass connections are commercially available for use with ₁₆¹-, ¹₈-, and ¹₄-in.-o.d. packed columns. The properties and characteristics of common types are presented in Table 3.12.

3.7.4 Filling the Column

The following procedure for packing columns, with practice, can produce the desired goal of a tight packing bed with minimum particle fracturing. First, a metal column is precoiled for easy attachment to the injector and detector of the instrument in which it is to be installed or a precoiled glass column configured for a specific instrument is procured from a vendor. Insert a large wad of glass wool partially into one end of the column, align the excess wool along the outside of the tubing, overlap the excess wool with vacuum tubing, and attach the other end of the vacuum tubing to a faucet aspirator or pump (12). After securing a small funnel to the other end of the column, add packing material in small incremental amounts into the funnel and gently tap the packing bed while applying suction.

After the column is completely packed, insert a small piece of silanized glass wool into the inlet end of the column, disconnect the vacuum, and remove the

TABLE 3.12 Ferrule Materials for Packed Columns

Material

Temperature

Limit (^◦C) Properties

Metal

Brass 250 Permanent connection on metal columns

Stainless steel 450 Permanent connection on metal columns Teflon 250 Low upper temperature limit and cold-flow

properties renders this material unsuitable for temperature programming and elevated temp. operation, reusable to some extent Ceramic-filled 250 Isothermal use only; conforms easily to glass;

used for connections to mass spectrometers Graphite (G) 450 High temperature limit with no bleed or

decomposition; soft and easily deformed upon compression; may be resealed only a limited number of times

Vespel 100%

polyimide (PI)

350 Good reusability factor, can be used with glass, metal and Teflon columns; may seize on metal and glass columns with use at elevated temperature

Vespel/graphite 85%

PI, 15% G

400 Excellent reusability; will not seize to glass or metal; performs better than graphite and Vespel alone; 60% PI composite seals with lesser torque and has added lubricity

vacuum tubing and the large wad of glass wool, replacing it with a smaller plug of silanized wool. This approach eliminates the exasperating sight of your packing material zipping out of the column during filling if a insufficiently tight wad of silanized glass wool was initially inserted into the outlet end of the column.

3.7.5 Conditioning the Column and Column Care

Before a column is used for analyses, it must be thermally conditioned by heat- ing the column overnight at an oven temperature below the upper limit of the stationary phase with a normal flowrate of carrier gas. The column should not be connected to the detector during conditioning. The purpose of conditioning is the removal from the column residual volatiles and low-boiling species present in the stationary phase, which otherwise would produce an unsteady baseline at elevated column temperatures, commonly referred to as “column bleed,” and contaminate the detector. Conditioning a column also helps in the redistribution of the liquid phase on the solid support. The degree of conditioning is dependent on the nature and amount of liquid phase in the column; usually heating a column overnight at an appropriate elevated temperature produces a steady baseline under chromato- graphic conditions the following day. Analyses using the more sensitive detectors (ECD, NPD, MS) may require an even longer column conditioning period.

The following guidelines can prolong the lifetime of a column:

1. Any gas chromatographic column, new or conditioned, packed or capillary, should be purged with dry carrier gas for 15–30 min before heating to a final elevated temperature to remove the detrimental presence of air.

2. A column should not be rapidly or ballistically heated to an elevated temperature but should be heated by slow to moderate temperature pro- gramming to the desired final temperature.

3. Excessively high conditioning and operating temperatures reduce the life- time of any gas chromatographic column.

4. Use “dry” carrier gas or install a moisture trap in the carrier-gas line. Do not inject aqueous sample on a column containing a stationary phase intolerant of water.

5. The accumulation of high-boiling compounds from repetitive sample injec- tions occurs at the inlet end of the column and results in discoloration of the packing. It is a simple matter to remove the discolored segment of packing and replace it with fresh packing material. This action prolongs the column lifetime.

6. Do not thermally shock a column by disconnecting it while it is hot. Allow the column to cool to ambient temperature prior to disconnection. Packings are susceptible to oxidation when hot.

7. Cap the ends of a column for storage to prevent air and dust particles from entering the column. Save the box in which a glass column was shipped for safe storage of the column.