《美国FDA农残分析手册》（第一卷英文版）Chapter 5 GLC

SECTION 502 Pesticide Analytical Manual Vol I Figure 502-b of columns can be based on such GLC Column Parameters parameters. More detailed discus- sion of parameters and conditions affecting each are found in any basic chromatography text, such as those listed in Section 505. Bibli ography Nonretained Retention Time. The most basic on time, the time ample introduction and elution of the analyte, measured at the peak maximum (t in Figure 502- b). Retention time is corrected for Column parameters are calculated from measurements the time required for a non- on a chromatogram produced by the column retained solute to reach the de- tector(tm), often called dead time r holdup time Corrected retention time (t'r) is the difference between t and For practical convenience, the peak caused by the solvent is used as the nonretair solute in pesticide residue determinations. Analyte retention time depends on the extent to which the analyte is retained by the particular stationary phase under a given set of conditions. Retention time is constant when column temperature and carrier gas flow are constant, so this characteristic is the GlC measurement that serves to identify the analyte; it can be measured electronically in seconds or manually in mm from the resultir chromatogram. Retention time measured from injection to peak maximum is often called“ absolute retention time.” Absolute retention time is affected by many column conditions that can vary including amount of liquid phase, temperature, carrier gas flow rate, column ducible to ist in tables of data intended to assist an analyst in ide cient repro- Instead,"relative retention times" are calculated and listed, because they are far more reproducible from day to day and among different instruments or labo- ratories Relative retention time (rrt)of an analyte is the ratio of its corrected retention time (t'r) to the corrected retention time of a"marker"(reference)compound The pesticide chlorpyrifos, molecular formula CqHuCi3nO,PS, is used in this manual as the marker compound for most systems, because it chromatographs well and contains all the heteroatoms to which selective GlC detectors respone tention times relative to chlorpyrifos(rrt for many pesticides and related co pounds are listed in Appendix I, PESTDATA For the same (or equivalent) liquid phase, rrt of an analyte is independent of column ty pe(packed us capillary), liquid load, column length, or carrier gas flow rate change. The rrts for a particular liquid phase vary significantly only with column temperature; rrt s in Appendix I are valid only at the temperature speci d for each column Capacity Factor Capacity factor describes the retentive behavior of a sample com- ponent relative to the retentive behavior"of a nonretained component. The 50a-4 Form FDA 2905a (6/92]

SECTION 502 Pesticide Analytical Manual Vol. I 502–4 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) of columns can be based on such parameters. More detailed discus￾sion of parameters and conditions affecting each are found in any basic chromatography text, such as those listed in Section 505, Bibli￾ography. Retention Time. The most basic measurement in chromatography is retention time, the time between sample introduction and elution of the analyte, measured at the peak maximum (tr in Figure 502- b). Retention time is corrected for the time required for a non￾retained solute to reach the de￾tector (tm), often called dead time or holdup time. Corrected retention time (t′ r ) is the difference between tr and tm. For practical convenience, the peak caused by the solvent is used as the nonretained solute in pesticide residue determinations. Analyte retention time depends on the extent to which the analyte is retained by the particular stationary phase under a given set of conditions. Retention time is constant when column temperature and carrier gas flow are constant, so this characteristic is the GLC measurement that serves to identify the analyte; it can be measured electronically in seconds or manually in mm from the resulting chromatogram. Retention time measured from injection to peak maximum is often called “absolute retention time.” Absolute retention time is affected by many column conditions that can vary, including amount of liquid phase, temperature, carrier gas flow rate, column length, and system volume. Thus, absolute retention times are insufficiently repro￾ducible to list in tables of data intended to assist an analyst in identifying analytes. Instead, “relative retention times” are calculated and listed, because they are far more reproducible from day to day and among different instruments or labo￾ratories. Relative retention time (rrt) of an analyte is the ratio of its corrected retention time (t′ r) to the corrected retention time of a “marker” (reference) compound. The pesticide chlorpyrifos, molecular formula C9H11C13NO3PS, is used in this manual as the marker compound for most systems, because it chromatographs well and contains all the heteroatoms to which selective GLC detectors respond; retention times relative to chlorpyrifos (rrtc ) for many pesticides and related com￾pounds are listed in Appendix I, PESTDATA. For the same (or equivalent) liquid phase, rrt of an analyte is independent of column type (packed vs capillary), liquid load, column length, or carrier gas flow rate change. The rrts for a particular liquid phase vary significantly only with column temperature; rrtcs in Appendix I are valid only at the temperature speci￾fied for each column. Capacity Factor. Capacity factor describes the retentive behavior of a sample com￾ponent relative to the “retentive behavior” of a nonretained component. The Figure 502-b GLC Column Parameters Column parameters are calculated from measurements on a chromatogram produced by the column. Inject Nonretained solute tm t′ r tr W

Pesticide Analytical Manual Vol. I SECTION 502 capacity factor of an analyte depends only on the time the analyte spends in the stationary phase, which is, chromatographically speaking, far more important than the time spent in the mobile phase. The capacity factor(k)of an analyte is cal- culated from analyte retention time as k =(t-t)/t Capacity factor should not be confused with"sample capacity, which describes the maximum amount(e. g, 50 ng) of an analyte that can be injected onto a chromatograph before column overload occurs. Column sample capacity depends on percent liquid load in packed columns and on column id and film thickness in capillary columns.) Selectivity. Stationary phase selectivity is simply defined as the ability of a phase to differentiate between analytes in the same injection The selectivity term is technically not interchangeable with polarity [1]. a polar column may exhibit very poor selectivity for a particular chemical species. In general, nonpolar stationary phases exhibit greatest selectivity for nonpolar analytes, and polar stationary phases exhibit greatest selectivity for polar analytes. Selectivity of a GlC system is defined synonymous with separation factor, relative retention, and selectivity factor and. g by both the stationary phase and the analytes In the literature, selectivity(a) calculated as kB/ka, where kB and ka are capacity factors of two adjacent peaks. In this calculation, a is always 21.0, but a separation factor of 1.0 indicates that no separation is possible in that system [2] Resolution. Resolution is the degree of separation between two chromatograph peaks and is related to time(capacity factor), selectivity, and efficiency. Consider- able information about resolution and its related parameters is available in general textbooks on chromatography. For practical purposes, however, it is enough to know that optimizing selectivity by choice of stationary phase will optimize resolu- tion. Despite the importance of column efficiency in analyzing complex samples, especially at low levels, increasing efficiency will not solve all separation problems and often will only increase analysis time. a different choice of stationary phase may solve a resolution problem more easily than a longer column will Resolution is considered optimized when calculated k values range between 2-10 Efficiency. In qualitative terms, column efficiency refers to the degree to which injected analyte is able to travel through the column in a narrow band. Visually, a more efficient col produces narrower, sharper peaks on the chromatogram The more efficient the column, the better able it is to resolve analytes that elute close to one another. Greater efficiency results in greater signal-to-noise ratio and hence increases sensitivity. Efficiency is measured quantitatively by calculating ical plates according to the formul N=16(RT/w) where n= total theoretical plates, RT=absolute retention time in mm,and width of peak base in mm, measured as the distance at the baseline between lines draw peak. The analyte te on calculated must be specified, because comparisons are only valid fc analytes eluting at the same absolute retention time. Column efficiency can also be expressed as height equivalent of one theoretical plate(HETP), i.e., column length(cm)/N; using this expression, smaller numbers represent more efficient columns. Calculation of theoretical plates/column length permits comparisons of different length columns m FDA 2905a(6/92 502-5

Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 502–5 Pesticide Analytical Manual Vol. I SECTION 502 capacity factor of an analyte depends only on the time the analyte spends in the stationary phase, which is, chromatographically speaking, far more important than the time spent in the mobile phase. The capacity factor (k) of an analyte is cal￾culated from analyte retention time as k = (tr -tm)/tm. (Capacity factor should not be confused with “sample capacity,” which describes the maximum amount (e.g., 50 ng) of an analyte that can be injected onto a chromatograph before column overload occurs. Column sample capacity depends on percent liquid load in packed columns and on column id and film thickness in capillary columns.) Selectivity. Stationary phase selectivity is simply defined as the ability of a phase to differentiate between analytes in the same injection. The selectivity term is technically not interchangeable with polarity [1]. A polar column may exhibit very poor selectivity for a particular chemical species. In general, nonpolar stationary phases exhibit greatest selectivity for nonpolar analytes, and polar stationary phases exhibit greatest selectivity for polar analytes. Selectivity of a GLC system is defined by both the stationary phase and the analytes. In the literature, selectivity (α) is synonymous with separation factor, relative retention, and selectivity factor and is calculated as kB/kA, where kB and kA are capacity factors of two adjacent peaks. In this calculation, a is always ≥1.0, but a separation factor of 1.0 indicates that no separation is possible in that system [2]. Resolution. Resolution is the degree of separation between two chromatographic peaks and is related to time (capacity factor), selectivity, and efficiency. Consider￾able information about resolution and its related parameters is available in general textbooks on chromatography. For practical purposes, however, it is enough to know that optimizing selectivity by choice of stationary phase will optimize resolu￾tion. Despite the importance of column efficiency in analyzing complex samples, especially at low levels, increasing efficiency will not solve all separation problems and often will only increase analysis time. A different choice of stationary phase may solve a resolution problem more easily than a longer column will. Resolution is considered optimized when calculated k values range between 2-10. Efficiency. In qualitative terms, column efficiency refers to the degree to which injected analyte is able to travel through the column in a narrow band. Visually, a more efficient column produces narrower, sharper peaks on the chromatogram. The more efficient the column, the better able it is to resolve analytes that elute close to one another. Greater efficiency results in greater signal-to-noise ratio and hence increases sensitivity. Efficiency is measured quantitatively by calculating theoretical plates according to the formula N = 16 (RT/w)2 where N = total theoretical plates, RT = absolute retention time in mm, and w = width of peak base in mm, measured as the distance at the baseline between lines drawn tangent to the two sides of the peak. The analyte on which theoretical plates are calculated must be specified, because comparisons are only valid for analytes eluting at the same absolute retention time. Column efficiency can also be expressed as height equivalent of one theoretical plate (HETP), i.e., column length (cm)/N; using this expression, smaller numbers represent more efficient columns. Calculation of theoretical plates/column length permits comparisons of different length columns

SECTION 502 Pesticide Analytical Manual Vol I Basic GlC texts, such as those listed in Section 505, provide additional explana tions about theoretical plate measurements, qualitative effect of column efficiency on peak shape, and practical means of improving peak shape. Column efficiency is referred to in this chapter when dis ussing relative advantages of diffe erent types of columns 502 B: PACKED COLUMNS During most of the over 30 years of GlC use in pesticide residue determination packed column GlC prevailed as the only practical option. During early develop- ment of open tubular capillary columns, when only traditional capillaries were available, packed columns offered distinct advantages in ease of use and capacity for injection of larger volumes of extract. Current availability of wide bore capil- lary columns has reversed the trend, however, and use of packed columns is diminishing Packed columns still offer advantages in ease of installation; no additional inlet adapters or other specialized hardware are needed to install packed columns into chromatographs designed for packed column operation. Packed columns can also still withstand repeated injections of extract better than capillary columns. How- ever, recent improvements in inlet systems and operating parameters for wide bore columns have increased their capacity for injected extract. Combined with the innately greater efficiency and inertness of wide bore columns, these improve- ments are encouraging the shift from packed to wide bore columns for routine use Components of Packed Columns Packed columns consist of packing material made by coating inert solid support with a thin film of stationary liquid phase, glass or metal tubing to contain the packing material, and silanized glass wool plugs used to hold the packing material in place within the tubing Solid Support. The solid support in packed GLC columns provides a large inert surface onto which the stationary liquid phase is deposited as a relatively uniform thin film. Solid support should provide as large a surface area as possible and should interact as little as possible with analytes. Desirable properties of solid supports are large surface area per unit volume, chemical inertness at high tem- peratures, mechanical strength, thermal stability, ability to be wetted uniformly by a stationary liquid phase, and ability to hold a liquid phase strongl The most frequently used solid supports for GLC column packings are derived from diatomaceous earth The structure of the diatomaceous earth consists essen- tially of three-dimensional lattices containing silicon with active hydroxyl and oxide groups on the surface. Untreated diatomaceous earth has considerable surface activity that must be reduced before it becomes a suitable support material. Sev eral techniques have been used to deactivate the surface activity of diatomaceous earth. Most frequently, the diatomaceous earth is acid-washed and then silanized with an agent such as dimethyldichlorosilane Different commercially available solid supports and even different lots of the same support may have different surface areas or variations ward lar analytes. Unpredictable behavior among solid supports provided the impetus 50a-6 Form FDA 2905a (6/92]

SECTION 502 Pesticide Analytical Manual Vol. I 502–6 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Basic GLC texts, such as those listed in Section 505, provide additional explana￾tions about theoretical plate measurements, qualitative effect of column efficiency on peak shape, and practical means of improving peak shape. Column efficiency is referred to in this chapter when discussing relative advantages of different types of columns. 502 B: PACKED COLUMNS During most of the over 30 years of GLC use in pesticide residue determination, packed column GLC prevailed as the only practical option. During early develop￾ment of open tubular capillary columns, when only traditional capillaries were available, packed columns offered distinct advantages in ease of use and capacity for injection of larger volumes of extract. Current availability of wide bore capil￾lary columns has reversed the trend, however, and use of packed columns is diminishing. Packed columns still offer advantages in ease of installation; no additional inlet adapters or other specialized hardware are needed to install packed columns into chromatographs designed for packed column operation. Packed columns can also still withstand repeated injections of extract better than capillary columns. How￾ever, recent improvements in inlet systems and operating parameters for wide bore columns have increased their capacity for injected extract. Combined with the innately greater efficiency and inertness of wide bore columns, these improve￾ments are encouraging the shift from packed to wide bore columns for routine use. Components of Packed Columns Packed columns consist of packing material made by coating inert solid support with a thin film of stationary liquid phase, glass or metal tubing to contain the packing material, and silanized glass wool plugs used to hold the packing material in place within the tubing. Solid Support. The solid support in packed GLC columns provides a large inert surface onto which the stationary liquid phase is deposited as a relatively uniform thin film. Solid support should provide as large a surface area as possible and should interact as little as possible with analytes. Desirable properties of solid supports are large surface area per unit volume, chemical inertness at high tem￾peratures, mechanical strength, thermal stability, ability to be wetted uniformly by a stationary liquid phase, and ability to hold a liquid phase strongly. The most frequently used solid supports for GLC column packings are derived from diatomaceous earth. The structure of the diatomaceous earth consists essen￾tially of three-dimensional lattices containing silicon with active hydroxyl and oxide groups on the surface. Untreated diatomaceous earth has considerable surface activity that must be reduced before it becomes a suitable support material. Sev￾eral techniques have been used to deactivate the surface activity of diatomaceous earth. Most frequently, the diatomaceous earth is acid-washed and then silanized with an agent such as dimethyldichlorosilane. Different commercially available solid supports and even different lots of the same support may have different surface areas or variations in inertness toward particu￾lar analytes. Unpredictable behavior among solid supports provided the impetus

Pesticide Analytical Manual Vol. I SECTION 502 for most laboratories to purchase precoated packing. Variations in solid support activity are of greatest concern when determining pesticide residues that are dif ficult to chromatograph, because such analytes are easily adsorbed or degraded during chromatography Adsorption or degradation of an analyte on a poor qual- ity solid support can affect the relative retention time of the analyte and the size and shape of the resulting peak. The most inert solid support material available should always be used to prepare column packings Chromatographic solid supports are available in a variety of mesh sizes. A support material of 80/100 mesh contains particles that will pass through an 80-mesh screen but not through a 100-mesh screen. Experiments have shown that column efficiency improves as solid support mesh number increases(particle size decreases) [3]. However, to maintain the same gas flow through a column, carrier gas pres- sure must be increased as solid support particle size decreases. Mesh size of 100/ 120 was shown to produce optimum efficiency for a 6 column of 4 mm id Col- umns 4-6 long and 2-4 mm id, filled with colum packings prepared from 80/100 or 100/120 mesh solid supports, are routinely used for residue determination Liquid Phase Load. No matter what liquid stationary phase is used, liquid load influences column efficiency and capacity(amount of sample extract that can be injected onto the col ). Packing materials with loads ranging from <l to 5% are routinely used for pesticide residue determination Liquid phase load can be varied without changing relative retention times of compounds if the same column temperature is used. At the same column tempera- ure and gas flow, a column with less liquid phase will allow compounds to elute more quickly than a higher load column. Carrier gas flow can be lowered when using columns with less liquid phase to permit compounds to elute at approxi- mately the same time as from higher load columns operated at higher gas flows Laboratory observations indicate that compounds with a tendency to degrade on pad is used, probably because the lower load is incapable of covering al hase or be adsorbed by a column are more likely to do so when a lower liquid phase support active sites. In these cases, analyte retention time and peak size will be affected, as described above. Residue analysts should be aware of the pitfalls of low load columns when dealing with compounds that are easily degraded or adsorbed Column Tubing Almost all columns used in pesticide residue determinations are made from glass tubing. Although some gas chromatographs require metal col- umns,so many problems occur with metal that they should be avoided. In the ast, new glass columns had to be cleaned and silanized in the laboratory to remove any residual caustic materials and to deactivate the column. today, most glass columns are silanized by the manufacturer and are purchased ready to use Inadequate deactivation of glass columns can cause peak tailing due to adsorption or degradation of the sample or standard on the active sites of the column itself. Glass Wool. Glass wool for use in GlC columns must be silanized to prevent adsorption; presilanized glass wool is available cially silanization can be performed by the laboratory. a plug of silanized glass wool is always used at the outlet(detector) end of a packed column to hold the packing material in place. Glass wool can also be used in the inlet end of a packed column but opinions vary about the advisability of this practice mittal No.94101/94 m FDA 2905a(6/92 502-7

Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) 502–7 Pesticide Analytical Manual Vol. I SECTION 502 for most laboratories to purchase precoated packing. Variations in solid support activity are of greatest concern when determining pesticide residues that are dif￾ficult to chromatograph, because such analytes are easily adsorbed or degraded during chromatography. Adsorption or degradation of an analyte on a poor qual￾ity solid support can affect the relative retention time of the analyte and the size and shape of the resulting peak. The most inert solid support material available should always be used to prepare column packings. Chromatographic solid supports are available in a variety of mesh sizes. A support material of 80/100 mesh contains particles that will pass through an 80-mesh screen but not through a 100-mesh screen. Experiments have shown that column efficiency improves as solid support mesh number increases (particle size decreases) [3]. However, to maintain the same gas flow through a column, carrier gas pres￾sure must be increased as solid support particle size decreases. Mesh size of 100/ 120 was shown to produce optimum efficiency for a 6' column of 4 mm id. Col￾umns 4-6' long and 2-4 mm id, filled with column packings prepared from 80/100 or 100/120 mesh solid supports, are routinely used for residue determination. Liquid Phase Load. No matter what liquid stationary phase is used, liquid load influences column efficiency and capacity (amount of sample extract that can be injected onto the column). Packing materials with loads ranging from <1 to 5% are routinely used for pesticide residue determination. Liquid phase load can be varied without changing relative retention times of compounds if the same column temperature is used. At the same column tempera￾ture and gas flow, a column with less liquid phase will allow compounds to elute more quickly than a higher load column. Carrier gas flow can be lowered when using columns with less liquid phase to permit compounds to elute at approxi￾mately the same time as from higher load columns operated at higher gas flows. Laboratory observations indicate that compounds with a tendency to degrade on or be adsorbed by a column are more likely to do so when a lower liquid phase load is used, probably because the lower load is incapable of covering all solid support active sites. In these cases, analyte retention time and peak size will be affected, as described above. Residue analysts should be aware of the pitfalls of low load columns when dealing with compounds that are easily degraded or adsorbed. Column Tubing. Almost all columns used in pesticide residue determinations are made from glass tubing. Although some gas chromatographs require metal col￾umns, so many problems occur with metal that they should be avoided. In the past, new glass columns had to be cleaned and silanized in the laboratory to remove any residual caustic materials and to deactivate the column. Today, most glass columns are silanized by the manufacturer and are purchased ready to use. Inadequate deactivation of glass columns can cause peak tailing due to adsorption or degradation of the sample or standard on the active sites of the column itself. Glass Wool. Glass wool for use in GLC columns must be silanized to prevent compound adsorption; presilanized glass wool is available commercially or silanization can be performed by the laboratory. A plug of silanized glass wool is always used at the outlet (detector) end of a packed column to hold the packing material in place. Glass wool can also be used in the inlet end of a packed column, but opinions vary about the advisability of this practice

SECTION 502 Pesticide Analytical Manual Vol I Used in the inlet end of a packed column, glass wool can cause adsorption or degradation of certain sensitive compounds. Problems with normally stable com- pounds can also occur when deposits of sample co-extractives collect on the glass In particular, when deposits of fatty extracts accumulate at the top of the colum analytes in subsequent injections can be partially trapped; errors in resid uantitation result. Elimination of glass wool at the inlet end of the column pears to minimize this problem by allowing injected co-extractives to spread over a portion of the column where subsequent analytes cannot be trapped so readily In other cases, glass wool in the inlet end of the column may prevent the rapid deterioration of columns caused by injecting co-extractives from fatty foods or other commodities that are difficult to clean up. Co-extractives trapped on the glass wool plug can be eliminated by replacing the plug, an easier, quicker, and less expensive process than replacing the packing material Choosing whether to use glass wool in the inlet end of the column appears to depend on several factors, including type of packing material used, commodit being analyzed, analytes of interest, type of detector, and method of analysis. Experience will dictate when the advantages of glass wool in the column inlet outweigh the disadvantages; a laboratory attempting to locate the source of prob lems in a glC determination should definitely investigate the effects of glass wool n the column inlet Preparation of Packed Columns Acceptable techniques for packing empty GlC columns are designed to fill the ith as much packing material as possible(i.e, to pack the material as tightly as possible) while breaking the fewest particles. Column efficiency increases with the amount of properly coated support in the column, and adsorption and degradation problems are minimized when careful handling of the packing mate. rial creates the fewest broken(active) sites Poor packing technique causes visible differences in column performance(eff ciency) and peak symmetry. Loosely packed columns or columns containing too little column packing are inefficient and a cause of inadequate separations On the other hand, a column packed too tightly requires excessive carrier gas pres- sure, which can result in the column becoming plugged with broken particles To pack a glass column Insert about 1-2"silanized glass wool into detector end of column, far enough from end to prevent packing material from extending into detec. or base where temperatures are usually much higher than column oper ating temperature · Use rubber tubing to ct detector end of column to (aspirator or vacuum pump); attach funnel with short piece of rubber tubing to inlet end of column Apply partial vacuum at detector end of column, and slowly add prepared packing material through funnel 50a-8 Form FDA 2905a (6/92]

SECTION 502 Pesticide Analytical Manual Vol. I 502–8 Transmittal No. 94-1 (1/94) Form FDA 2905a (6/92) Used in the inlet end of a packed column, glass wool can cause adsorption or degradation of certain sensitive compounds. Problems with normally stable com￾pounds can also occur when deposits of sample co-extractives collect on the glass wool. In particular, when deposits of fatty extracts accumulate at the top of the column, analytes in subsequent injections can be partially trapped; errors in residue quantitation result. Elimination of glass wool at the inlet end of the column ap￾pears to minimize this problem by allowing injected co-extractives to spread over a portion of the column where subsequent analytes cannot be trapped so readily. In other cases, glass wool in the inlet end of the column may prevent the rapid deterioration of columns caused by injecting co-extractives from fatty foods or other commodities that are difficult to clean up. Co-extractives trapped on the glass wool plug can be eliminated by replacing the plug, an easier, quicker, and less expensive process than replacing the packing material. Choosing whether to use glass wool in the inlet end of the column appears to depend on several factors, including type of packing material used, commodity being analyzed, analytes of interest, type of detector, and method of analysis. Experience will dictate when the advantages of glass wool in the column inlet outweigh the disadvantages; a laboratory attempting to locate the source of prob￾lems in a GLC determination should definitely investigate the effects of glass wool in the column inlet. Preparation of Packed Columns Acceptable techniques for packing empty GLC columns are designed to fill the column with as much packing material as possible (i.e., to pack the material as tightly as possible) while breaking the fewest particles. Column efficiency increases with the amount of properly coated support in the column, and adsorption and degradation problems are minimized when careful handling of the packing mate￾rial creates the fewest broken (active) sites. Poor packing technique causes visible differences in column performance (effi￾ciency) and peak symmetry. Loosely packed columns or columns containing too little column packing are inefficient and a cause of inadequate separations. On the other hand, a column packed too tightly requires excessive carrier gas pres￾sure, which can result in the column becoming plugged with broken particles. To pack a glass column: • Insert about 1-2" silanized glass wool into detector end of column, far enough from end to prevent packing material from extending into detec￾tor base where temperatures are usually much higher than column oper￾ating temperature. • Use rubber tubing to connect detector end of column to vacuum source (aspirator or vacuum pump); attach funnel with short piece of rubber tubing to inlet end of column. • Apply partial vacuum at detector end of column, and slowly add prepared packing material through funnel