ASTM E260 Standard Practice for Packed Column Gas Chromatography
8. Hazards
8.1 Gas Handling Safety - The safe handling of compressed gases and cryogenic liquids for use in chromatography is the responsibility of every laboratory. The Compressed Gas Association, a member group of specialty and bulk gas suppliers, publishes the following guidlines to assist the laboratory chemist to establish a safe work environment: CGA P-1, CGA G-5.4, CGA P-9, CGA V-7, CGA P-12, and HB-3.

9. Preparation of Packed Gas Chromatographic Columns
9.1 Preparation of the Tubing Material:
9.1.1 Glass columns should be cleaned and deactivated, first by rinsing with 30 mL acetone and then 30 mL toluene. Next, fill the column with 10 volume % solution of dimethyldichlorosilane in toluene. Allow the solution to stand in the column for 30 min. Finally, rinse the column with anhydrous toluene and then anhydrous methanol to cap unreacted DMDCS CL groups. Dry the column by passing a stream of dry nitrogen through it. Cap both ends of the column until such time that it can be packed.

9.1.2 Metal columns should be cleaned thoroughly before packing by rinsing with methanol, acetone, and chloroform. The column should be dried by passing nitrogen or dry air through it. Do not blow house air through the column since this compressed air usually contains an oil aerosol from the pump.

NOTE 3 - Most chromatographic supply houses provide metal tubing that has been washed with solvents and is ready for use.

9.1.3 An alternative procedure is recommended for nickel tubing and can be used to clean stainless steel tubing. Rinse the nickel tubing with ethyl acetate, methanol, and distilled water. Then fill the tube with 20 volume % nitric acid and let it stand for 10 min. (Warning: Work in a hood and wear safety equipment when using nitric acid.) Next, rinse the tube with distilled water to neutrality and then rinse with methanol and acetone. Finally, dry the column by blowing nitrogen or helium through it.

9.1.4 The column length is generally 3 to 6 ft (1 to 2 m). Shorter columns can be used to decrease the time of analysis or to separate high boiling compounds. Longer columns are used to improve resolution, but have longer analysis times. (Columns longer than 20 ft (6.1 m) require excessive pressures to maintain the proper carrier gas flow.) A compromise is usually made between analysis time and resolution. As a general rule, an increase or decrease of column length by a factor of 3 to 4 is necessary to see a significant change in peak separation.

9.1.5 The diameter of the column can be 1/8 in. (3.2 mm) or 1/4 in. (6.4 mm) outside diameter. The 1/8-in. column has less sample capacity, but greater efficiency, and is the most common type. Glass columns are generally 2 mm or 4 mm inside diameter. Some analysts have found that 3/16 in. (4.8 mm outside diameter) metal columns are the ideal combination between the capacity of 1/4 in. (6.4 mm outside diameter) columns and the efficiency of 1/8 in. (3.2 mm) outside diameter columns.

9.2 Choice of Diatomaceous Earth Support for Packed Columns - See 7.3.

9.3 Phase Loading on Diatomaceous Supports - For preparative work and analysis of substances boiling below room temperature, use 15 wt % loadings for white-type supports and 30 wt % for pink-type supports. For general work, use loadings of the range of 3 to 15 wt %. For highest efficiency, shortest retention times, and the least amount of bleed during high-temperature operation, use 3 wt. % loadings. The lower phase loadings have lower sample capacity and elute components more rapidly and at lower temperatures. Always check the manufacturers' literature for suggested phase loadings for a particular support. For some applications (especially headspace analysis) loadings as low as 0.2 wt. % are used which result in very narrow peaks and short analysis times. High phase loadings tend to produce less reactive packings.

9.4 Preparation of the Gas Chromatographic Packing - The following procedures describe the coating of a solid support with stationary phase. The following four methods are commonly used to prepare gas chromatographic packings: (a) Filtration or Solution Coating Method, (b) Rotating Evaporator Method (c) Evaporative Method, and (d) Vacuum Evaporative Method. When preparing packings with loadings in the range of less than 5 wt %, the Filtration or Solution Coating Method is recommended. This method is preferred because it provides minimum handling of the friable white-type supports. For loadings of more than 5 wt. %, other methods can be used. The Rotating Evaporator Method is recommended, but should only be used if a rotating evaporator is available, which turns very slowly at 20 to 30 rev per min.

NOTE 4 - A 5 wt. % loading of stationary phase consists of 5 g stationary phase added to 95 g of support.

9.4.1 Filtration or Solution Coating Method - Prepare 100 mL of a solution of the desired phase in a vacuum filter flask. Use a suitable high boiling solvent (boiling point more than 60°C). The actual loading of the liquid phase on the support will depend upon both the viscosity of the phase solution and the density and mesh size of the support.
9.4.1.1 Add 20 g of support to the filter flask. Reduce the pressure in the filter flask for a few minutes with a water aspirator, then release the vacuum. Repeat this procedure for several cycles in order to remove air bubbles from the pores of the support particles. Be prepared to release the vacuum if the slurry foams excessively.

9.4.1.2 Allow the slurry to stand for several minutes. Pour the slurry into a coarse-frit sintered-glass filter funnel, and allow the solvent to drain freely until the support settles.

9.4.1.3 Apply vacuum cautiously and stop instantly when the solvent stops dripping. Dump the support into a flat borosilicate glass dish, and allow it to dry. Do not scrape the particles out of the funnel, since this might crush the particles. Do not resieve before use.

9.4.1.4 The actual phase loading will depend upon the viscosity of the phase solution and both the density and particle size of the support. For example, a 2 % solution of dimethyl silicone gum liquid phase will give a 3.8 wt % loading on white-type supports. A10 wt % solution of a less viscous liquid phase will give a 5.5 wt % loading on white-type supports and 7.5 wt % on pink-type supports. Loadings obtained with other phases on other supports are best determined by experimentation.

9.4.1.5 The best way to determine the percent loading is to extract it from the support by extraction in a Soxhlet apparatus and determine the weight loss. Alternatively, measure the volume of solution recovered and calculate the volume of solution held up by the support. Calculate the approximate percent loading on the support by assuming that the concentration of the solution does not change.

9.4.2 Evaporative Method:
9.4.2.1 Weigh out the desired amounts of support and phase. Use a larger amount than that required to account for attrition, spills, etc. Dissolve the liquid phase in a chemically inert, low-boiling solvent contained in a filtration flask (see Table 2). (Most catalogs of gas chromatography equipment suppliers contain lists of suitable solvents.)

9.4.2.2 Gradually add the support to the solution with gentle swirling or agitation but with no mechanical stirring. (Suggested solvents are given in Table 2.) The amount of solution should be just enough to wet the solid support and form a slurry with little excess solvent.

9.4.2.3 Evacuate the flask briefly several times to remove air bubbles from the pores of the support. Be prepared to release the vacuum if the slurry foams excessively.

9.4.2.4 Transfer the slurry to a large flat borosilicate glass dish, and slowly evaporate the solvent in a hood with no further handling. The dish must be of a size that the packing is spread on the bottom in a thin layer, no more than about 1/4-in. thick. A borosilicate glass baking dish makes a suitable container.

9.4.2.5 The critical stage occurs when excess solvent has evaporated, but the bed is still quite damp with a slight excess of solvent. Break up the damp bed by gently raking it with a spatula. As the solvent evaporates from the surface of a static bed of support, it leaves a higher concentration of phase at the bed surface. Therefore, the bed must be broken up frequently during the final stages of solvent evaporation to prevent formation of an unevenly coated support.

9.4.2.6 Continue to air-dry the material in the hood until the last traces of solvent are gone. Avoid excessive handling of the particles to prevent formation of fines due to abrasion, especially in the case of the white-type supports.

9.4.3 Rotating Evaporator Method - Prepare the slurry of support and phase as described in 9.4.2.1 to 9.4.2.3, except in an indented, round-bottom flask. Connect the flask to a rotating evaporator. Rotate the flask very slowly (less than 20 to 30 revolutions per minute) and evaporate the solvent under reduced pressure (water aspirator). Very slow rotation is necessary to prevent the particles from abrading against each other. Use of a heat lamp increases the evaporation rate. This method is not recommended for fluorocarbon supports.

9.4.4 Vacuum Evaporative Method - Prepare a slurry of support and phase in a filtration flask of sufficient capacity. (Suitable solvents are given in Table 2.) Attach the flask to a vacuum source (water aspirator) and apply vacuum briefly. (Be prepared to release the vacuum if the slurry foams excessively.) Repeat this procedure several times in order to remove the air bubbles from the pores in the support.
9.4.4.1 Apply the vacuum for a longer period, and swirl the contents of the flask occasionally until all the solvent is almost evaporated. This is the critical stage.

9.4.4.2 Now shake the contents of the flask by gently bumping the flask on a wood or plastic board. This will break up the bed of packing. Do not allow the solvent to evaporate from the surface of the support bed. Otherwise, the solvent will evaporate and leave a higher concentration of phase at the bed surface.

9.4.4.3 Continue to apply vacuum until the packing is a freely flowing powder, then transfer it to a tray for air-drying in a hood.

9.4.5 Fluidized Drying Technique - This technique has been used to produce efficient, uniformly coated packings. During the drying stages of methods 9.4.1 to 9.4.4, when the packing has reached the consistency of a wet sand, add it to a fluidizer. Then dry the packing by passing a flow of inert, warmed gas (nitrogen or helium) through the bed of packing.

9.5 Packing the Gas Chromatographic Column - The purpose in packing a gas chromatographic column is to fill the column with packing as completely as possible, leaving no empty spaces. Two variations are noted in 9.5.3 and 9.5.4 (a pressure-fill procedure and a vacuum fill procedure).
9.5.1 It is preferable to coil the column before packing to prevent crushing of the support particles. Metal columns can be coiled after loading to meet equipment requirements. Bends in the packed region must never be made with radii less than those specified in 9.5.2, to avoid crushing the packing in the column.

9.5.2 Right-angle bends are often necessary to make connections to injection and detection systems, and must be made before packing the column since some tubing deformation will occur, which will crush some of the solid support. Bends for such purposes should be within 4 in. (10 cm) of the column ends. For coiled columns, minimum diameter mandrels should be as follows: for 1/8 in. (3.2 mm) OD column use a 1 1/2-in. (38-mm) mandrel; for 1/4 in. (6.4 mm) OD column use a 2-in. (51-mm) mandrel. These configurations do not preclude the use of U- or W-shaped columns. If a U- or W-shaped column is to be used, the minimum 180° bend diameter must be at least that given for the above mandrel sizes.

9.5.3 Pressure Fill Procedure - To each end of the column to be filled, fit a nut, a back ferrule, and a suitable front ferrule. Place a small plug of silanized glass wool into the detector end of the column, and cap the column by screwing in a metal cap with a 1/16-in. vent hole drilled into it. When analyzing trace acidic compounds, as organic acids and phenols, adsorption can be decreased by using phosphoric acid-treated glass wool to plug the column ends. Wear safety glasses when pressure-packing columns.
9.5.3.1 Attach the end of the empty column to an apparatus similar to that shown in Fig. 2. Add to the reservoir sufficient packing material to fill the column, plus about 30 %. Attach the upper end of the reservoir to a nitrogen supply line controlled to provide approximately 40 psi. Check that all connections are tightened, place a safety shield in front of the setup, and apply 40 psi to the system.

9.5.3.2 As the stationary phase starts to fill the column, gently tap the column with a wood rod (handle of spatula or screwdriver) or an electrical vibrator set at a very low vibration level. Continue tapping until the packing shows no voids and the level of packing in the reservoir remains constant.

9.5.3.3 Shut off the nitrogen supply and wait for the pressure to dissipate. Disconnect the column from the reservoir. Do not disconnect the column while it is under pressure. Have a clean beaker available to collect excess packing material that will fall from the opened reservoir. Tap out about 1/8 in. (3 mm) of column packing, and replace it with a silanized glass wool plug. Affix a metal column tag engraved with a description of the stationary phase, loading, support, and the assigned column number.

9.5.4 Vacuum-Fill Procedure:
9.5.4.1 Clamp the column so that the detector and injector ends point upward. Plug the detector end of the column with a 1/4-in. plug of silanized glass wool. Use phosphoric-acid treated glass wool when analyzing for trace organic acids and phenols.

9.5.4.2 Attach a small funnel to the injection port end of the column. Attach the detector end of the column to a vacuum source, either a vacuum pump (preferably) or a water aspirator. (If a water aspirator is used, a 500-mL filter flask, or the device shown in Fig. 3, should be placed in the line between the pump and the column.) Do not turn on the vacuum yet.

9.5.4.3 Add 1 to 2 mL of packing to the funnel, and tap the column gently to settle the packing. A pencil or a wooden spatula handle can be used. Alternatively, the column can be stroked with a plastic saw. The use of an electric vibrator is not recommended. Excessive vibration will cause the particles to abrade against each other, producing fines and newly fractured surfaces that are not coated with stationary phase.

9.5.4.4 Turn on the vacuum source. Continue to add the packing in small increments with tapping until the column is full. Finally, apply pressure to the head of the column to pack it a little tighter. However, take care to make sure the pressure is equalized slowly, because packing will be blown out of the column if the pressure is released too suddenly.

9.5.4.5 Next, tap out enough packing to create a 1/8 in. (3 mm) void space at the injector port end of the column. Plug this end with a silanized glass wool plug. Do not pack the plug too tightly. This will either impede the carrier gas flow or crush the packing particles.

9.5.4.6 Higher efficiencies are always observed if the column is packed for on-column injection. In this technique, the column is packed so that there is space at the injection port end of the column, which is then placed inside the injection port. This void space should be of such a length that the injection needle just reaches, or slightly penetrates, only the glass wool plug, not the packing, when the column is installed. Thus the sample is injected almost directly onto the column.

9.6 Conditioning of Packed GC Columns:
9.6.1 The purpose of the conditioning process is to remove extraneous material (solvent and adsorbed material) from the column before analytical usage. Since the column is heated, the liquid phase also redistributes itself over the support surface to provide a more even coating.

9.6.2 Install the column into the gas chromatograph at the injection side only. Do not connect the column to the detector during the conditioning stage. Any column bleed might foul the detector and the connection lines between the column and detector. Turn on the normal analytical carrier gas flow and flush air out of the column at ambient temperature for 30 min.

9.6.3 Heat the column at a rate of 2°C/min to the conditioning temperature. The latter temperature should be at least 25°C higher than the analytical temperature but 25°C lower than the maximum operating temperature recommended for the liquid phase. Maintain this temperature overnight with carrier gas flow.

9.6.4 The next day cool the column and connect it to the detector. Detectors operated in very sensitive modes, particularly the electron capture detector, might require two or more days of conditioning at the higher temperatures before a satisfactory baseline is obtained. (Other sources for baseline drift and noise are impurities in the carrier gas, a dirty detector, air leaks in the gas-line fittings, insufficient carrier gas pressure, a much-punctured septum, chemical decomposition of the phase (due to presence of traces of acid or base on the support, in the phase, or on the inner column walls, and incorrect fuel gas ratios to the flame ionization detector.)

9.6.5 There is a special "no-flow" conditioning procedure which can be used with certain silicone phases, as methyl and methylphenyl silicones with or without low vinyl content. It has been reported to improve analysis for drug compounds. The procedure starts by conditioning the column for 1/2 h as described in 9.6.2. Turn off the carrier gas flow, cap the free end of the column with a metal cap, and heat at 310°C for 1.5 h with no carrier gas flow. Cool the column to 100°C. Uncap the oven, turn on the carrier gas flow, and continue the regular conditioning procedures listed in paragraph 7.6.3.

NOTE 5 - This no flow conditioning procedure may damage or destroy non-silicone containing stationary phase or silicone containing functional groups other than phenyl or methyl.

9.6.6 Many of the liquid phases are commercial-grade material, and conditioning might require several days before the noise level is low enough to provide usable baselines at high sensitivity. The use of gas chromatographic-grade phases is recommended since they have been carefully purified and long periods of conditioning are usually not necessary.