Theory and Instrumentation of GC. GC Columns

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1 Theory and Instrumentation of GC GC Columns i Wherever you see this symbol, it is important to access the on-line course as there is interactive material that cannot be fully shown in this reference manual.

2 Aims and Objectives Aims and Objectives Aims To compare and contrast Packed and Capillary columns for GC To give a revision session of fundamental intermolecular interactions and relate the various types of interaction to retention in GC To explore the various stationary phase types and explain the critical factors in choosing a phase To investigate important physical parameters of capillary GC columns and their relationship with retention, resolution and efficiency is GC separations Describe column bleed and how to minimise practically Highlight good practice for column installation and conditioning Objectives At the end of this Section you should be able to: Recognise and explain the various interactions that occur between analytes and stationary phases in GC Select an appropriate stationary phase for various analyte and application types Select appropriate column dimensions for various application types Demonstrate a good understanding of how to manipulate column physical parameters in order to obtain good quality separations in a reasonable timeframe Explain the principles of hood column handling, installation and conditioning in a practical context

3 Content Open Tubular Capillary Columns 3 Deactivation 4 Comparison of Packed and Capillary GC columns 4 Characteristics of packed and capillary WCOT columns 5 Analyte & Stationary Phase Polarity 6 Electronegativity 7 Dispersive Interactions 8 Dipole Interactions 9 Hydrogen Bonding 10 Stationary Phases 10 Polysiloxanes 10 Polyethylene Glycols 15 Stationary Phase Selection 16 General Considerations 16 Dispersive Interactions 17 Dispersive Interactions and Polarity 18 Dipole Interactions and Hydrogen Bonding 20 Stationary Phase Selection 23 PLOT Columns 23 Applications 24 Summary 26 Stationary Phases for Packed Column GC 27 Stationary Phase 27 Support Particle 28 Column Dimensions 29 Length 29 Internal Diameter 31 Film Thickness (d f ) 34 Phase Ratio (β) 36 Carrier Gas Flow Rate 38 Column Bleed 39 Bonded and Cross-linked Stationary Phases 40 Column temperature limits 40 Measuring bleed 41 Column Installation & Conditioning 41 Column Cutting 42 Column Installation 43 Preparing the column 43 Importance of column cuts 44 Conditioning 44 Confirm Installation 45 Crawford Scientific 2

4 Open Tubular Capillary Columns Capillary columns were first used and patented by Golay in 1958/9, but were not widely used until the late 1970 s, after which they have steadily grown in popularity. Today the vast majority of GC applications are developed or run using capillary columns. Open Tubular capillary columns consist of a long narrow tube of silica coated on the inside surface with a very thin film of immobilised polymeric liquid, gum, particulate or zeolite stationary phase. These columns are known as Wall Coated Open tubular columns (WCOT) and provide the highest resolution and efficiency of all gas chromatographic columns. This is mainly due to the length and homogeneously thin stationary phase films that can be achieved with capillary column technology. Fused silica is the most popular material of construction for WCOT (capillary) columns as it is flexible, inert and produces very high efficiency columns as the polymeric stationary phases wet the silica surface well to produce homogeneous films. Even though the silica used has high tensile strength, the very thin silica tubing (~25 µm) is susceptible to rapid corrosion and breakage in a laboratory atmosphere. Therefore the columns are coated with a sheath of polyimide, which provides a protective layer and allows a maximum operating temperature (for the silica column) of around 360 o C. Typical cross section of a Wall Coated Open Tubular (WCOT) capillary column Section of a 30m capillary column on a hangar (frame) to allow to use with a reasonable sized GC oven. For applications requiring higher oven temperatures, stainless steel clad silica columns are used. The inner surface of fused silica tubing is chemically treated to minimize interactions of the sample with the tubing. The reagents and process used depend on the type of stationary phase being coated onto the tubing. A silylation process is used for most columns. Silanol groups (Si-OH) on the tubing surface are reacted with a silane type reagent. Crawford Scientific 3

5 Deactivation A silylation process is used for most columns. Silanol groups (Si-OH) on the tubing surface are reacted with a silane type of reagent. Typically, a methyl or phenyl-methyl silyl surface is created for most columns. Typical deactivation reaction using trimethylchlorosilane to reduce the activity (polarity) of the surface silica silanol groups. Comparison of Packed and Capillary GC columns Packed GC columns are typically manufactured from glass or stainless steel. They are packed with pellicular silica particles (typically between 30/40 mesh and 100/120 mesh) onto which the stationary phase is coated. The smaller the particle size the higher the column efficiency. A typical packed GC column will be between 2 and 4 meters in length with an internal diameter of between 2 and 4 mm. Since capillary WCOT columns are open tubular, the pressure drop (backpressure created) across the column is very low, and therefore they can be manufactured in lengths of 60m+, which would be impossible using packed column technology. Capillary GC columns are manufactured with thin uniform liquid phase films, generating high efficiency usually in the order of 3,000 5,000 plates per meter. Packed column stationary phase films are thicker and less uniform - generating a much lower efficiency in the order of 1,000 to 2,000 plates per meter. The total available plates / column for capillary columns are therefore much higher than for packed columns as is shown in the Performance Comparison opposite. Packed columns also have a band broadening contribution due to Eddy Diffusion a van Deemter term that leads to reduced efficiency. Of course as capillary columns are not packed with particulate material, this term is eliminated from the band broadening contribution, Golay described this situation in his adaptation to the Van Deemter equation. Crawford Scientific 4

6 Typical capillary GC column configuration: 30m 0.25mm i.d. 0.1μm film thickness. Characteristics of packed and capillary WCOT columns The inner diameter (I.D.) of capillary columns ranges from about 0.18 mm to 0.53mm. The I.D. of packed columns ranges from 2 to 4 mm. The separation power of the respective columns is given by the total chromatographic plate counts. These values are calculated for typical lengths of 20 meters for a capillary and 2 meters for a packed column. Comparing the total chromatographic plates shows that the capillary column have from times the separating power as packed columns. If we compare column efficiencies, by normalizing the chromatographic efficiency to the length of the column (i.e., plates/m), we see that capillary columns are more efficient than packed columns. The high separating power of capillary columns is primarily due to the column length and thin stationary phase films that can be achieved by this column technology. There is a trade off for attaining this high separating power. Column capacity (i.e., the mass of individual solutes that can be injected into the columns in nanograms (ng)), for very narrow bore 0.18 capillary columns is limited to about 100 ng per component and increases to ng for megabore (0.53 mm) columns. By comparison, the capacity for packed columns is considerably higher, on the order of 10,000 ng. Table 1. Performance comparison Properties Column I.D. (mm) Capacity (ng) ,000-2,000 10,000 He flow (ml/min) Plates/m 5,300 3,300 1,600 2,500 Total plates 159,000 99,000 48,000 5,000 Chromatograms Crawford Scientific 5

7 Arachlor 1260 is a commercial blend of polychlorinated biphenyl materials. The upper chromatogram was acquired using a typical packed GC column (2m x 2mm i.d.) using electron capture detection (ECD). The chromatogram shows an efficiency of around 1500 plates and around 16 discrete peaks are observed. The lower chromatogram shows the same sample chromatographed using a capillary GC column (50m x 0.25mm i.d.). The sample was split 30:1 to reduce the sample loading on column. Here we see over 65 separate components, in a shorter analysis time and the column generates 150,000 plates. Analyte & Stationary Phase Polarity Before we begin to investigate stationary phase types and their interaction with analyte molecules, it s important to understand the concept of molecular polarity and dipole interactions. These interactions form the basis of the fundamental adsorption mechanisms that cause analyte retention in GC. We also classify GC stationary phase types according to their polarity (non-polarity) and so a good understanding is very important. All covalently bonded molecules will share electrons between the bonded atoms. A nonpolar covalent bond has a uniform distribution of electron charges between the atoms the simplest non-polar covalent bonds exist in homonuclear diatomic species such as Cl 2 or H 2. In this type of molecule there is no permanent localised electrical charge build up, electrons are shared uniformly within the molecule. Alternatively, a polar bond displays a non-uniform electron distribution cloud. This typically occurs when two non-metal atoms which are more than two positions apart in the periodic table are involved in the bond some examples are shown below. Crawford Scientific 6

8 The blue arrow head shown under the molecule indicates the direction of highest negative charge and displays that the molecule has a dipole moment, i.e. it contains a relatively positive and negative centre (it is polarised). The dipole moments on some other common organic molecules is also shown opposite. Where there is more than one dipole (polar bond) within the molecule it will also have an Overall Dipole Moment indicating the overall charge polarisation. Electronegativity Electronegativity is an index that relates the relative attraction an element has for electrons within a covalent bond. As can be seen from the diagram above electronegativity follows the same trends as the atomic radii. It is possible to predict the presence of a dipole moment within a molecule by comparing the electronegativity of the bonded atoms. C-H = = 0.4 EN units difference therefore the bond will be non-polar. C-0 = = 1.0 EN units difference therefore the bond will be polar. Crawford Scientific 7

9 Some other typical bonds of interest in general analytical chemistry: C-F = = 1.5 EN units difference (Very polar bond around the limit at which covalent and ionic bonds are differentiated). C-N = = 0.5 EN units difference (Polar Bond) 0-H = = 1.4 EN units difference (Very polar bond) C-S = = 0.0 EN units difference (Non-Polar Bond). Dispersive Interactions In GC, retention of solute molecules occurs due to stronger interaction with the stationary phase than the mobile phase. In GC, the situation is unique in that the chemical interaction with the mobile phase is very small indeed therefore the interactions between the analyte molecules and the stationary phase are of great importance this concept will be discussed further. In GC the interaction between the analyte and stationary phase can be divided into three braid categories these are: Dispersive interactions Dipole Interactions Hydrogen Bonding Dispersive Interaction Dispersive interactions are the most difficult to describe and visualise, as they are caused by charge fluctuations that occur throughout a molecule that arise from electron/nuclei vibrations. The fluctuations are random in nature and are basically a statistical effect. Every molecule has a number of arrangements of nuclei and electrons having dipole moments that fluctuate resulting in an overall molecular charge of zero. However, at any instant in time, the dipoles are capable of interacting with other instantaneous dipoles of other molecules. Dispersive forces are ubiquitous and must arise in all molecular interactions. They can, themselves, occur in isolation, but are always present even when other types of interaction dominate. An example of interactions that are exclusively dispersive are those between hydrocarbons. The lower molecular weight hydrocarbons are liquids and not gasses due entirely to the dispersion forces that act between the hydrocarbon molecules (to access the complete explanation, please refer to the online material, the visualisation of the phenomena get easier through the interactive presented material). i Crawford Scientific 8

10 Dipole Interactions There are two distinctive classes of dipole-dipole interaction, those between two species containing a permanent dipole (dipole-dipole interactions) and those between a molecule possessing a permanent dipole and a polarisable molecule (dipole-induced dipole interactions). Dipole-dipole interactions can be very strong and occur between molecules with permanent dipoles, examples of this type of interaction occur between alcohols, esters, ethers, amines, amides, nitriles. Of course, as well as the dipole interaction there will also be a contribution to the intermolecular attraction from the dispersive interaction, however, the strength of the dipole-dipole interaction will far exceed any dispersive interactions that occur. Dipole-induced dipole interactions occur when a molecule containing a permanent dipole approaches a molecule that is polarisable, most commonly these molecules would contain π-electron systems (i.e. aromatic or unsaturated compounds). The strength of this interaction lies between dispersive and dipole-dipole interactions. Again these interactions will occur alongside any purely dispersive interaction that occurs between the molecules (to access the complete explanation, please refer to the online material, the visualisation of the phenomena get easier through the interactive presented material). i Crawford Scientific 9

11 Hydrogen Bonding Hydrogen bonding is a special case of a dipole-dipole interaction in which the dipoles associated with (most usually) hydroxyl groups of the two molecules come into close proximity. Hydrogen bonding interactions are very strong compared to dispersive interactions and in the extreme (e.g., the association of water with methanol) the dipoledipole interaction energy can approach that of a chemical bond. Some examples of hydrogen bonding are shown opposite. It should be noted that even when molecules are undergoing hydrogen bonding there is still an underlying weak dispersive interaction occurring simultaneously. Table 2. Typical energy of interaction Interaction Energy (kj/mol) Dispersive <<1 Dipole-Induced Dipole 1 Dipole-Dipole 3.3 Hydrogen bonding 19 Stationary Phases Polysiloxanes Polysiloxanes are the most common stationary phases. They are available in the greatest variety and are the most stable, robust and versatile. Standard polysiloxanes are characterized by the repeating siloxane backbone. Each silicon atom contains two functional groups. The type and amount of the groups distinguish each stationary phase and its properties. The most basic polysiloxane is the 100% methyl substituted. When other groups are present, the amount is indicated as the percent of the total number of groups. For example, a 5% diphenyl-95% dimethyl polysiloxane contains 5% phenyl groups and 95% methyl groups. The "di-" prefix indicates that each silicon atom contains two of that particular group. Sometimes this prefix is omitted even though two identical groups are present for example this phase is sometimes known as 5% Phenyl. If the methyl percentage is not stated, it is understood to be present in the amount necessary to make 100% (e.g., 50% phenyl-methyl polysiloxane contains 50% methyl substitution). Crawford Scientific 10

12 Cyanopropylphenyl percent values can be misleading. A 14% cyanopropylphenyldimethyl polysiloxane contains 7% cyanopropyl and 7% phenyl (along with 86% methyl). The cyanopropyl and phenyl groups are on the same silicon atom, thus their amounts are summed. Methyl Polysiloxane Phases 100% dimethyl polysiloxane. Dominant Interaction types: Dispersive Functional group Dispersion Dipole Hydrogen bonding Methyl Strong None None Phase Names alternative phase names DB-1, BP-1, SPB-1, CP-Sil 5, Rtx-1, OV-1, SE-30, 007-1, ZB-1 Phenyl Phases 5% di-phenyl dimethylpolysiloxane. Dominant Interaction types: Dispersive/Induced dipole. Functional group Dispersion Dipole Hydrogen bonding Phenyl Very strong Weak None Phase Names alternative phase names DB-5, SPB-5, XTI-5, Mtx-5, CP-Sil 8CB, SE-54, RTX-5, BPX-5, MDN-5, Rtx-5ms, BP5, ZB-5 Phenyl Phases 35% di-phenyl dimethylpolysiloxane. Crawford Scientific 11

13 Dominant Interaction types: Dispersive/Induced dipole. Functional group Dispersion Dipole Hydrogen bonding Phenyl Very strong Weak None Phase Names alternative phase names DB-35, RTX-35, SPB-35, AT-35, Sup-Herb, MDN-35, BPX-35 Phenyl Phases 50% di-phenyl dimethylpolysiloxane. Dominant Interaction types: Dispersive/Induced dipole. Functional group Dispersion Dipole Hydrogen bonding Phenyl Very strong Weak None Phase Names alternative phase names DB-17, RTX-50, CP-Sil 19, BPX-50, SP-2250 Cyanopropyl Phases 6% cyanopropylphenyl dimethylpolysiloxane. Dominant Interaction types: Dipole/Dispersion/Hydrogen bonding. Functional group Dispersion Dipole Hydrogen bonding Cyanopropyl Strong Very strong Moderate Phase Names alternative phase names DB-35, RTX-35, SPB-35, AT-35, Sup-Herb, MDN-35, BPX-35 Crawford Scientific 12

14 Cyanopropyl Phases 14% cyanopropylphenyl dimethylpolysiloxane. Dominant Interaction types: Dipole/Dispersion/Hydrogen bonding. Functional group Dispersion Dipole Hydrogen bonding Cyanopropyl Strong Very strong Moderate Phase Names alternative phase names DB-1701, SPB-1701, CP-Sil 19 CB, Rtx-1701, CB-1701, OV-1701, , BPX-10. Cyanopropyl Phases 50% cyanopropylphenyl dimethylpolysiloxane. Dominant Interaction types: Dipole/Dispersion/Hydrogen bonding. Functional group Dispersion Dipole Hydrogen bonding Cyanopropyl Strong Very strong Moderate Phase Names alternative phase names DB-23, Rtx-2330, , SP-2330/2340/2380/2560 Crawford Scientific 13

15 Cyanopropyl Phases 50% cyanopropylmethyl dimethylpolysiloxane. Dominant Interaction types: Dipole/Dispersion/Hydrogen bonding. Functional group Dispersion Dipole Hydrogen bonding Cyanopropyl Strong Very strong Moderate Phase Names alternative phase names DB-23, Rtx-2330, , SP-2330/2340/2380/2560 Trifluoropropyl Phases 35% trifluoropropyl dimethylpolysiloxane. Dominant Interaction types: Dipole/Dispersion/Hydrogen bonding. Functional group Dispersion Dipole Hydrogen bonding Trifluoropropyl Strong Moderate Weak Phase Names alternative phase names DB-200, Rtx-200 Trifluoropropyl Phases 50% trifluoropropyl dimethylpolysiloxane. Dominant Interaction types: Dipole/Dispersion/Hydrogen bonding. Functional group Dispersion Dipole Hydrogen bonding Trifluoropropyl Strong Moderate Weak Phase Names alternative phase names DB-210 Crawford Scientific 14

16 Table 3. Overview Functional Dispersion Dipole Hydrogen bonding group Methyl Strong None None Phenyl Very strong Weak None Cyanopropyl Strong Very strong Moderate Trifluoropropyl Strong Moderate Weak Polyethylene Glycols Polyethylene glycols (PEG) are widely used as stationary phases. Stationary phases with "wax" or "FFAP" in their name are polyethylene glycol or a derivative. Standard polyethylene glycol stationary phases are not substituted, thus the polymer is 100% of the stated material. They are less stable, less robust and have lower temperature limits than most polysiloxanes. With typical use, they exhibit shorter lifetimes and are more susceptible to damage upon over heating or exposure to oxygen. The unique separation properties of polyethylene glycol makes these liabilities tolerable. Polyethylene glycol stationary phases must be liquids under GC temperature conditions. There are two types of polyethylene glycols in common use as GC stationary phases. One has a higher upper temperature limit, but exhibits slightly higher activity (i.e., peak tailing for some compounds) and is known an Extended Temperature Range Wax. The other has a lower temperature limits but exhibits better reproducibility and inertness, these are the standard Wax phases. The separation characteristics of the two stationary phases are slightly different. Another variation of polyethylene glycol phases are ph modifications. Free Fatty Acid Phase (FFAP) columns are terephthalic acid modified polyethylene glycols. These columns are used for the analysis of acidic compounds. Base modified polyethylene glycol stationary phases are also available for the analysis of basic compounds. Strong acids and bases often exhibit peak tailing for standard columns. ph modified stationary phases may decrease the amount of tailing for strong acids or bases. O OH n Typical Structure of Polyethylene Glycol (PEG) Phases Dominant Interaction types: Dispersive. Table 4. Overview Functional group Dispersion Dipole Hydrogen bonding Methyl Strong None None Phenyl Very strong Weak None Cyanopropyl Strong Very strong Moderate Trifluoropropyl Strong Moderate Weak PEG Strong Strong Moderate Crawford Scientific 15

17 Phase Names alternative phase names DB-Innowax, BP-20, 007-CW, CP-WAX 52 CB, Stabilwax, Supelcowax-10, DB-Wax, Rt- Wax Base modified PEG Phases: CAM, Carbowax Amine, Stabilwax-DB, CP-51 WAX Acid modified PEG Phases: DB-FFAP, OV-351, SP-1000, Stabilwax-DA, 007-FFAP, Nukol Stationary Phase Selection The selection of the correct stationary phase is one of the most critical parameters in the success of any GC method. As the interaction of the analyte molecules with the mobile phase is almost negligible, the column temperature and the interaction of the analyte with the stationary phase will govern the selectivity of the separation. The use of temperature will be discussed further. The following discussion highlights the important considerations in choosing an appropriate stationary phase for GC separations. General Considerations In choosing an appropriate GC stationary phase it is generally accepted that the principle of like dissolves like holds well, and that to separate polar analytes a polar stationary phase is required, and vice versa. The skill of stationary phase selection lies is knowing (or empirically discovering), the degree of polarity required to avoid overly long retention times whilst still obtaining a satisfactory separation. Further, when separating compounds of intermediate polarity or where the analytes are a mixture of polar and non-polar compounds, further knowledge of the retentivity and selectivity of each phase is required. We shall base our discussion here on the three main mechanisms of interaction namely Dispersion, Dipole (including dipole - induced dipole) and Hydrogen Bonding. Non-polar test compounds (n-alkanes) chromatographed on a non-polar (100% diemthyl polysiloxane) column. Non-polar test compounds (n-alkanes) chromatographed on a polar (carbowax) column note the lack of retention and poor peak shape. Like Dissolves Like. Non-polar analyte interacting with a non-polar stationary phase. Crawford Scientific 16

18 Polar analyte interacting with a polar stationary phase. Dispersive Interactions All common stationary phase types are capable of interacting with analyte molecules via a dispersive mechanism with the methyl and phenyl polysiloxanes show the strongest interaction. Dispersive interactions can be correlated with analyte volatility, in general the more volatile an analyte the less retained it will be and the earlier it will elute from a column with a predominantly dispersive stationary phase. Analyte boiling point can be used as an indicator of analyte volatility therefore analytes with low boiling points will elute quickly from columns with a strongly dispersive stationary phase. Whilst the boiling point prediction works well for analytes of the same functional chemistry (homologous series are an ideal example), when the analyte chemistry varies, the boiling point analogy may not work so effectively. Note that the boiling point analogy fails for compounds with alternative functionality to the alkyl-benzenes and n-alkanes (i.e. benzaldehyde, aniline and phenol). Column DB1, 30m x 0.25mm, 0.25µm. Carrier gas Helium 32 cm/sec, temperature o C (5 o C/min) Note that the boiling point analogy holds well for the n-alkanes, with the relative spacing of the peaks reflecting the difference in analyte boiling point. When the aromatic species are added to the sample even though the compounds elute in boiling point order, the relative peak spacing is lost due to differences in analyte chemistry. Crawford Scientific 17

19 Dispersive Interaction between non-polar stationary phase (100% dimethyl polysiloxane) and dispersive analytes (n-ocatne and ethytlbenzene). When dealing with analytes with mixed functionality the boiling point analogy is often confounded as can be seen. Note that s-butylbenzene and n-decane have the same boiling point and are separated via the differing strengths of their dispersive interaction. Alkanes chromatographed on a a non-polar stationary phase (DB1, 30m x 0.25mm, 0.25µm). Carrier gas Helium 32 cm/sec, temperature o C (5 o C/min) Note that the boiling point analogy fails for compounds with alternative functionality to the alkyl-benzenes and n-alkanes (i.e. benzaldehyde, aniline and phenol). As a general rule of thumb, if the boiling point of two compounds differs by 30 o C or more, then they may be separated by most stationary phases. This is due to the fact that dispersion is the dominant interaction for a wide range of stationary phase types. If the compounds boiling point differs by less than 10 o C (and the compounds do not belong to a homologous series), then the boiling point analogy is more likely to be in error. Dispersive Interactions and Polarity Another stationary phase characteristic that may affect retention in a predictable manner is the phenyl content. The higher the phenyl content of the stationary phase, the higher will be the retention of aromatic solutes RELATIVE to aliphatic solutes. This does not necessarily mean that the aromatics are more retained but that they shift relative to aliphatic solutes. This is demonstrated in the top example opposite. Increasing the phenyl Crawford Scientific 18

20 content of the phase causes earlier elution of the n-alkanes and later elution of the alkylbenzenes. The polarity of the stationary phase is not directly related to its selectivity but it does affect the retention and thus separation. In other words, polar compounds are more strongly retained by polar stationary phases and vice versa. Altering the phenyl content of the stationary phase affects the polarity of the column. By increasing the phenyl content the polarity increases (due to the presence of p -electrons in the aromatic groups) which renders the stationary phase less non-polar. This is well described in the lower example where the retention of the polar analytes (alcohols) is increased relative to the non-polar analytes (n-alkanes) where the phenyl content of the phase (and hence the polarity) is increased. 1. Toluene, 2. n-octane, 3. Ethylbenzene, 4. n-nonane, 5. Propylbenzene, 6. sec- Butylbenzene, 7. n-decane, 8. 1,2-Dichlorobenzene, 9. Butylbenzene, 10. n-undecance, 11. n-dodecane The more polar substituted benzene analytes increase in retention time whilst the n- alkanes show a reduction in retention time, when switching from a less polar column (DB1) to a more polar column (DB35). 1. n-tridecane, 2. 1-Undecanol, 3. n-tetradecane, 4. 1-Dodecanol. The more polar alcohol analytes show increased retention whilst the n-alkane retention reduces when switching from a less polar column (DB1) to a more polar column (DB35). Crawford Scientific 19

21 DB1/DB35, 30m x 0.25mm, 0.25µm. Carrier gas Helium 32 cm/sec, temperature o C (5 o C/min) Dipole Interactions and Hydrogen Bonding If the stationary phase is capable of dipole interaction, it enhances its power to separate solutes whose dipole moments are different. Cynopropyl, trifluoropropyl and Poly Ethylene Glycol (PEG) phases all show good dipole interaction properties. The degree of peak separation for solutes with different dipoles often changes if a stationary phase with a different amount of the dipole interaction is used. If the dipole difference between compounds is small, a greater amount of the appropriate phase is required (i.e. 50% cyanopropylphenyl-methyl instead of 14% cyanopropylphenyl-methyl). It is difficult to be predictive about the magnitude of the separation change for all peaks. Empirical studies show that this type of phase are well suited for compounds which have a base or central structure to which different groups are attached in various positions. Examples include substituted aromatics, halocarbons, pesticides and drugs. The same stationary phases that undergo dipole interactions also undergo hydrogen bonding interactions with the PEG phases showing the strongest interaction. Again where the analyte hydrogen bonding potential differs only slightly, a stationary phase with a greater amount of the appropriate group is required. This is demonstrated opposite. When a trifluoropropyl stationary phase is used, the meta and para- Xylene peaks and meta- and para-cresol peaks begin to show some degree of seaparation, based entirely on the increase in hydrogen bonding capacity of the stationary phase. Crawford Scientific 20

22 1. p-xylene, 2. m-xylene, 3. o-xylene. Column DB1/DB200, 30m x 0.25mm, 0.25µm. Carrier gas Helium 32 cm/sec, temperature o C (5 o C/min) The difference in dipole moment between the meta- and para- substituted forms of Xylene is very small. Using a non-dipole interaction phase (DB1), there is no discernible separation between these two positional isomers. However, when a dipole component is introduced into the stationary phase (trifluoropropyl), the separation between the two forms begins to occur. It may be postulated that increasing to a 50% trifluoropropyl phase will improve the separation even further. Dipole-Dipole interactions between m-xylene and the trifluoropropyl stationary phase. Crawford Scientific 21

23 1. p-xylene, 2. m-xylene, 3. O-Xylene, 4. 1,3-Dichlorobenzene, 5. 1,4-Dichlorobenzene, 6. 1,2-Dichlorobenzene, 7. o-cresol, 8. p-cresol, 9. m-cresol. Column DB1/DB200, 30m x 0.25mm, 0.25µm. Carrier gas Helium 32 cm/sec, temperature o C (5 o C/min) Hydrogen Bonding interaction between m-cresol and the trifluoropropyl stationary phase. Table 5. Hydrogen Bonding Analytes Strength Compounds Strong Alcohols, carboxylic acids, amines. Moderate Aldehydes, esters, ketones. Weak to None Hydrocarbons, halocarbons, ethers. Crawford Scientific 22

24 Stationary Phase Selection PLOT Columns Porous Layer Open Tubular (PLOT) columns are intended for Gas Solid Chromatographic (GSC) applications. PLOT columns are capillaries in the conventional sense but the inner wall of the capillary is coated with small, solid porous particles using a binder. The particles are usually Alumina or Molecular sieve and solutes are separated on differences in their adsorption properties, with size and shape differentiation also occurring. PLOT columns are used primarily for the separation of highly volatile liquids and permanent gases without the need for cryogenic or subambient cooling of the GC oven. Separations that would require column temperatures well below ambient temperatures, even with thick film capillary columns, can be obtained at ambient temperatures or above using PLOT column technology. Alumina columns are well suited to the analysis of C 1 C 10 hydrocarbons and small aromatics, whilst the KCl derivatised version of the column produces altered selectivity for the same compound group. The Q designated columns show better selectivity for C 1 -C 3 hydrocarbons, but give very long retention and broadened peaks for anything heavier than C 6. These columns are also able to separate sulphur gases and most light hydrocarbons. Molecular sieve columns are used to separate many noble and permanent gas samples and are also good for the separation of solvents. Crawford Scientific 23

25 Where: Rt-Msieve 5A PLOT 30m, 0.32mm Sample: gas standard, 3 5% each minor component in helium Injection: 1.0µL split (split ratio 1:10), 4mm inlet liner Injection temperature: 30 C Carrier gas: hydrogen, constant pressure Linear velocity: 30 C Oven temp: 30 C, isothermal Det: 200 C Applications Sulphur gases 15 m x 0.32 mm (ID) GasPro GSC Column. Carrier: He at 30 cm/sec. Oven temperature: 25 C for 3 min (10 C/min). Detector: TCD Solvents 30m, 0.53mm ID Rt-QPLOT 70µL split injection of solvent mixture Sample conc: 1% each component Oven temp: 100 C to C/min. (hold 2 min.) Inj. & det. temp: 220 C Carrier gas: helium Linear velocity: 23.6cm/sec. 100 C FID sensitivity: 1.28 x AFS Split ratio: 7.7:1 Split: 44.5cc/min. Column flow: 6.6cc/min. Crawford Scientific 24

26 Polypropylene Impurities 50 m aluminum oxide Carrier: 29 cm/sec. Oven temperature: 40 C for 3 min (10 C/min) to 200 C. Hydrocarbon gases 30m, 0.32mm ID Rt-QPLOT MT. 30.0µL injection of hydrocarbon gas mix Sample conc.: mol% each component Oven temp.: 40 C to C/min. (hold 10 min.) Injector: split/250 C Carrier gas: helium (constant pressure mode) Head pressure: 18.0psi Column flow rate: 40 C Linear velocity: 40 C Split ratio: 20:1 Det.: FID/240 C Make-up gas flow: 45cc/min. Inlet liner: 4mm single gooseneck Crawford Scientific 25

27 Refinery gases. 50m, 0.53mm ID Rt-Alumuna MT PLOT. 100µL (gas-tigh syringe, split injection) hydrocarbon gas mix, 100ppm each component Oven temp: 40 C to C/min. (hold 5 min.) Inj. and det. Temp: 200 C Carrier gas: helium Linear velocity: 37.5cm/sec. 80 C (5.0 ml/min) Split flow: 60 ml/min Summary 1. If no information or ideas about which stationary phase to use is available, start with a DB-1 or DB Low bleed ("ms") columns are usually more inert and have higher temperature limits. 3. Use the least polar stationary phase that provides satisfactory resolution and analysis times. Non-polar stationary phases have superior lifetimes to polar phases. 4. Use a stationary phase with a polarity similar to that of the solutes. This approach works more times than not; however, the best stationary phase is not always found using this technique. 5. If poorly separated solutes possess different dipoles or hydrogen bonding strengths, change to a stationary phase with a different amount (not necessarily more) of the dipole or hydrogen bonding interaction. Other co-elutions may occur upon changing the stationary phase, thus the new stationary phase may not provide better overall resolution. 6. If possible, avoid using a stationary phase that contains a functionality that generates a large response with a selective detector. For example, cyanopropyl containing stationary phases exhibit a disproportionatly large baseline rise (due to column bleed) with NPDs. 7. A 100% Methyl or 5% Phenyl, 50% Phenyl, 14% Cyanopropylphenyl and WAX (PEG) cover the widest range of selectivities with the smallest number of columns. Crawford Scientific 26

28 8. PLOT columns are used for the analysis of gaseous samples at above ambient column temperatures. The following link is an excellent web-page on phase polarity and column selection for various applications. Stationary Phases for Packed Column GC We have previously described that packed GC columns contain a particulate adsorbent onto which the stationary phase is coated. Over the many years that packed column GC was the most popular (and only) option for GC columns over 1000 stationary phases and supports have been invented. There were many more stationary phase types than exist for capillary columns due to the inherent lack of efficiency of packed columns, making selectivity of the stationary phase more important. There are far too many phase and support types to list them all here so we will attempt to describe some of the most popular combinations and summarise the various categories. Stationary Phase There are literally thousands of stationary phase types for packed column GC as has been stated. It is impossible to discuss all types here. Stationary phases range from the non-polar Squalane polymethyl siloxane phases, through the intermediately polar Apiezon range of phases to the polar Waxes (such as Carbowax). The OV range of phases from the Ohio Valley company (Marietta, Ohio, USA) contains equivalents to most capillary GC phases. The web-links below provide a host of valuable information on packed column GC phases from various manufacturers: ns.pdf Crawford Scientific 27

29 Support Particle There have been a number of materials used as supports for packed GC columns including, Celite (a proprietary form of a diatomaceous earth), fire-brick (calcined Celite), fire-brick coated with metallic silver or gold, glass beads, Teflon chips and polymer beads. Today however, the vast majority of contemporary packed GLC columns are filled with materials that are either based on of Celtic or polystyrene beads as a support. There are also a variety of support treatments to deactivate the support by end-capping silanol groups or removing metal oxides ultimately resulting is an improved GC peak shape. Some of the more common support treatments are outline below: Table 6. Common support treatments Treatment Contents of the Treatment NAW Non acid washed AW Acid washed (neutral in ph) BT Base treated (alkaline in ph) BW Base washed (neutral in ph) AW-BT Acid washed and base treated (alkaline) AW-BW Acid and base washed (neutral) AW-DMCS Acid washed and DMCS treated (neutral) Some typical applications of packed column GC are presented below, however most GC methods are now developed using capillary column techniques and packed column GC continues to decline in popularity for the reasons stated here and in previous pages. 6ft x 1/8", 2.0mm ID SilcoSmooth tubing 5% Carbowax 20M on 80/120 CarboBlack B Inj: 500μL, headspace Conc: 0.05gm/dL Oven temp: 90 C Inj. & det. temp: 200 C Carrier gas: helium Flow rate: 18mL/min. Crawford Scientific 28

30 Volatiles from Scotch Whisky. 5% Carbowax 20M 80/120 CarboBlack B 2m, 1/8" OD x 2mm ID Silcosmooth tubing 0.5µL on-column injection. Oven temp: o C (4 o C/min) Inj./det. temp: 200 o C/250 o C Carrier gas: Nitrogen Flow rate: 20mL/min FID sensitivity: AFS Aromatics (Xylene Isomers / BTEX). 5% RT-1200/5% Bentone / 120 Silicoport TM 2m, 1/8 OD 2mm SilcoSmooth tubing. Sample size: 0.1 µl Conc: 0.5μg/μL in hexane Oven temp: 100 o C Inj. & det. temp: 200 o C Carrier gas: Nitrogen Flow rate: 20mL/min FID sensitivity: AFS Column Dimensions Length When choosing a capillary GC column, as well as the stationary phase type, the physical dimensions of the column must also be specified usually Length, Internal Diameter and Stationary Phase Film Thickness. All of these dimensions are critical to the performance of any separation and each will be considered here starting with column length. Column length affects three important parameters: Efficiency Retention (analysis time) Pressure Crawford Scientific 29

31 Column efficiency is proportional to column length doubling column length will double the number of available theoretical plates and hence double efficiency. However resolution is proportional to the square root of efficiency therefore doubling column length will only provide a theoretical increase of 41% in resolution (often only 20-30% increase is achieved in practice). Doubling the column length will double the analysis time for isothermal operation and increase analysis time by a factor of for gradient temperature programmed analysis. This therefore reduces the importance of increasing column length to obtain or improve a separation. In practice column length is only increased when peak separation is very small and high efficiency is required, or when the sample contains many analytes all of which need separating. Increasing the column length also increases the pressure required to achieve a set flow rate which is not a problem practically unless very narrow columns are used. Increasing the column length also increases the cost of the column! L vs t R curve Doubling column length increases analyte retention by a factor of for temperature-programmed methods. Efficiency Crawford Scientific 30

32 Resolution curve Resolution increases by a factor or 41% if efficiency is doubled note the limiting behaviour at high plate count! i Separation on RTX mm 1.0mm, 20 o C/min to 250 o C. Flow 200mL/min. Where: 1. Phenol 2. n-decane 3. 2-Chlorophenol 4. 2,4-Dimethylphenol 5. 2-Nitrophenol 6. 2,4-Dichlorophenol Analysis conditions. (Temperature Program) Column 5% Diphenyl methylpolysiloxane Temp Program 50 o C (no hold) / 20 o C per min. 250 o C Flow: 2mL/min (constant flow) Inlet Split 50:1 150 o C Detector FID 210 o C Internal Diameter Altering the column diameter affects five operational parameters efficiency, retention, carrier flow rate, capacity and pressure drop across the column. The column internal diameter is inversely proportional to column efficiency. Therefore, halving the column internal diameter doubles the efficiency and improves resolution by a theoretical factor of Increases in efficiency arise due to the increase in analyte / stationary phase interactions in the smaller diameter tubes. Crawford Scientific 31

33 Analyte retention is also inversely proportional to column internal diameter for isothermal separations but crucially the retention time change under temperature gradient conditions is times the original retention. This makes changing the column internal diameter much more attractive in practical terms. Column head pressure is approximately an inverse square function of column radius, i.e. a 0.25mm i.d. column requires 1.7 times greater head pressure than a 0.32mm i.d. column of the same length at the same temperature. Columns of 0.18 mm or greater are used routinely smaller diameter columns often require instrument configuration changes to achieve the very high pressures required. Short, wide columns (e.g. 15m x 0.32mm) are impractical for use with GC-MS systems as the vacuum at the column outlet greatly reduces the head pressures required often to an uncontrollably low level. Column capacity increases with column internal diameter the capacity also depends on the stationary phase type, film thickness and the nature of the analytes. See the table opposite for typical column capacities in a range of different column internal diameters. Efficiency Resolution curve ID vs t R curve Crawford Scientific 32

34 i Separation on RTX mm 1.0mm, 20 o C/min to 250 o C. Flow 200mL/min. Where: 1. Phenol 2. n-decane 3. Chlorophenol 4. 2,4-Dimethylphenol 5. 2-Nitrophenol 6. 2,4-Dichlorophenol 7. 4-Chloro 3-methylphenol Analysis conditions. (Temperature Program) Column 5% Diphenyl methylpolysiloxane Temp Program 50 o C (no hold) / 20 o C per min. 250 o C Flow: 2mL/min (constant flow) Inlet Split 50:1 150 o C Detector FID 210 o C Table 7. Efficiencies Column ID (mm) Theoretical Plates/meter (max for analyte k=5) , , , , , , ,240 Table 8. Column capacity (ng per analyte on column) Film thickness Column diameter (mm) (µm) , ,000-2, ,000-2,000 1,200-2,000 2,000-3,000 Crawford Scientific 33

35 Important 1. Capillary GC columns are typically avialbale in the following lengths (m): 10, 15, 20, 25, 30, 50, 60, Extending the column length is the least favoured option for increasing resolution and should be avoided if possible 3. Column efficiency (N) is proportional to column length 4. Doubling the column length increases the resolution by times 5. Use the shortest column that will give you the required resolution (begin with m columns is the best length is unknown) 6. To increase resolution try changing the stationary phase or column internal diameter first 7. Column cost is proportional to column length 8. Analysis time is proportional to column length 9. Column head pressure and bleed increase with column length m columns are well suited to samples containing well separated analytes or where the number of analytes is low m columns should be used only where very large numbers of components need to be separated and as a last resort when reducing the column internal diameter and changing the stationary phase and temperature program have failed! Film Thickness (d f ) Column stationary phase film thickness (d f ) affects five critical GC parameters these being: retention, inertness, capacity, resolution and bleed. Under isothermal conditions, film thickness is directly proportional to retention time (the proportionality is approximately 1.5:1 under temperature gradient conditions). Thick stationary phase films are used to gain retention for highly volatile analytes such as solvents or some selected permanent gases. Increasing the film thickness allows retention of volatile analytes at temperatures at or above ambient. Analytes have equal or greater retention at higher column temperatures. The same principles apply when reducing film thickness and in this way the retention of highly adsorbed analytes (late eluting high boiling point or high molecular weight analytes), may be reduced using thinner film columns. Early eluting analytes (k<2) are better resolved using thicker film columns. Resolution will also increase for most analytes with k values between 5 and 10, however analytes with k>10 will see no improvement in resolution when a thicker film is used. For any given stationary phase, thicker films will bleed more and the upper temperature limits of thick film columns will be lower than their thin film counterparts. Thicker film columns are more inert as the film shields the analyte from active sites on the silica tubing increasing film thickness can often improve the peak shape of tailing peaks in capillary GC. Thicker film columns have higher analyte capacity, and so may reduce peak broadening this is of special interest when one analyte is present in vast excess compared to the others. Thicker film columns may prevent co-elution with the larger peak. Crawford Scientific 34

36 Resolution curve (Resolution versus Film Thickness) Peak 2 i Peak 4 Peak 7 Capacity factor versus Film Thickness (d f ) Peak 2 Peak 4 i Peak 7 Crawford Scientific 35

37 Where: 1. Phenol 2. n-decane 3. 2-Chlorophenol 4. 2,4-Dimethylphenol 5. 2-Nitrophenol 6. 2,4-Dichlorophenol 7. 4-Chloro 3-methylphenol Analysis conditions. (Temperature Program) Column 5% Diphenyl methylpolysiloxane Temp Program 50 o C (no hold) / 20 o C per min. 250 o C Flow: 2mL/min (constant flow) Inlet Split 50:1 150 o C Detector FID 210 o C Phase Ratio (β) The phase ratio of a column is a measure of the stationary phase to mobile phase ratio at any point in the column and is calculated using equation 1 opposite. Increasing the phase ratio will result in decreased analyte retention this can be seen from equation 2. The phase ratio can be increased by increasing the column radius or decreasing the film thickness. The opposite is also true if the phase ratio is decreased, analyte retention time will increase. The phase ratio can be decreased by reducing the column internal diameter or increasing the film thickness. Note that decreasing the phase ratio will also result in an increase in column capacity. The real elegance of using phase ratio is that it can be used to keep retention time the approximately constant whilst altering other aspects of the chromatography. For example: if one wanted to increase the efficiency of a separation this can be achieved by reducing the column internal diameter however this leads to increased analysis time at constant pressure and temperature. However, by choosing a thinner film the phase ratio can be kept approximately constant the net result is a more efficient separation within the same timescale as the original separation! Crawford Scientific 36

38 =156 =160 The table next shows the phase ratio of most column radius and film thickness combinations as long as the phase ratio is kept approximately constant, column i.d. may be reduced to obtain more efficient separations in a similar timescale. Table 10. Phase ratio for selected column dimensions Film Thickness d f (μm) Column diameter (mm) ,125 1, Crawford Scientific 37

39 Carrier Gas Flow Rate We already discussed that different carrier gases produce the highest efficiency separations at different carrier gas flow rates. In practice, linear velocity is a better measure of the carrier gas speed than flow rate the average linear velocity (ū) can be thought of as the length of column travelled by a carrier gas molecule every second. The van Deemter curve opposite (top) highlights several important practical issues: Hydrogen and Helium produce the most efficient separations at the average linear velocities used in capillary GC (20-50 cm/sec.) In practice velocities much higher than the mimima can be used to shorten analysis time without compromising the separation quality (often called the optimum practical gas velocity OPGV) When using Hydrogen or Helium with capillary columns it is usual to have the carrier linear velocity between 20 and 40 cm/sec. Liner velocity is dependent on temperature as temperature changes the carrier gas viscosity changes causing the carrier speed (liner velocity) to change at constant pressure. Some instruments are equipped with electronic pressure control, which can alter column head pressure as temperature changes to maintain constant linear velocity or constant carrier gas flow. Typical values of column pressures and flows for various column dimensions with both hydrogen and helium are shown in the Typical Data table presented below. Table 11. Typical Data Column Length 40cm/sec. 20cm/sec. 10m 0.42 min min. 15m 0.63 min min. 20m 0.83 min min. 30m 1.25 min min. 45m 1.88 min min. 60m 2.50 min min. 75m 3.13 min min. 105m 4.38 min min. Column lenght ( cm) L Average linear velocity u Dead volume time(sec) Crawford Scientific 38 t M

40 An excellent linear velocity calculator can be found at: Column Bleed Column bleed is the elution of degradation products of the stationary phase causing a background signal in the GC detector. All columns produce bleed products and the most common degradation reaction is the 7 member so-called back biting reaction shown opposite. It should be noted that a rising baseline during a GC temperature program is often attributed solely to column bleed whereas this may also be caused by septum bleed products, sample matrix bleed, changes in the carrier flow rate (mass flow sensitive detectors only) etc. Bleed is best characterised using a bleed profile a temperature program that ramps to the column top operating temperature and holds for 10-15mins. A typical profile is shown opposite any major deviations from this type of profile are not due to column bleed. Bleed is a continuous process - any peaks in a blank run are not from column bleed, but most likely originate from contaminants in the GC system. Column bleed increases with stationary phase polarity, film thickness and column age. Exposing the column to oxygen (air) or consistently using the column at its upper temperature limit accelerates the onset of higher column bleed. A sudden or rapid increase in column bleed is usually an indicator of column damage or a problem in the GC system. Prolonged heating of a column above its upper temperature limit, constant exposure of the column to oxygen (usually via a leak), or repeated injection of damaging compounds (water, acetonitrile etc.) are the most common causes of problems. Important Column Bleed Characteristics 1. The baseline is relatively flat during the lower temperature region of the blank run 2. A sharp rise in the baseline begins at o C below the upper limit of the column and continues until the upper temperature limit is reached 3. Upon holding at the upper temperature limit, a fairly flat baseline is obtained. Several minutes may elapse before the baseline becomes completely flat. Major deviations from this profile are not due to column bleed 4. Column bleed is an abnormal elevated baseline at high temperature it is not an elevated baseline at low temperature, a wandering or drifting baseline or discrete peaks Crawford Scientific 39

41 Stationary phase degradation Bonded and Cross-linked Stationary Phases Cross-linked stationary phases have the individual polymer chains linked via covalent bonds. Bonded stationary phases are covalently bonded to the surface of the tubing. Both techniques impart enhanced thermal and solvent stability to the stationary phase. Also, columns with bonded and cross-linked stationary phases can be solvent rinsed to remove contaminants. Most polysiloxanes and polyethylene glycol stationary phases are bonded and cross-linked. Column temperature limits Consider for the 30m x 0.25mm x 0.25mm, -60 / 325 (350) o C Columns have lower and upper temperature limits (1-3 above). If a column is used below its lower temperature limit, broad peaks are obtained (i.e., loss of efficiency). No column damage will occurr; however, the column does not function properly. Using the column at or above its lower limit maintains good peak shapes. Upper temperature limits are often stated as two numbers. The lower one is the isothermal temperature limit. The column can be used indefinitely at this temperature and reasonable column bleed and lifetime are realized. The upper number is the temperature program limit. A column can be maintained at this temperature for minutes without severely shortening column lifetime or experiencing excessively high column bleed. Exposing the column to higher temperatures or for longer time periods results in higher column bleed and shorter column lifetimes. Exceeding the upper temperature limits may damage the stationary phase and the inertness of the fused silica tubing. Crawford Scientific 40

42 Measuring bleed Excessive column bleed appears as a larger rise in the baseline at the higher temperature regions. There is no absolute measurement to indicate when column bleed is excessive. Column bleed is best measured as the difference or change in the background signal at two temperatures Relative Bleed. Usually the column s upper temperature limit and a lower value around 100 o C are used. The absolute background signal is a composite of the background generated by the entire GC system. It is not possible to determine the contribution of column bleed to this total background signal. By measuring the relative amount of column bleed, the other contributors to the background signal are subtracted out. Most columns are tested using FIDs. The output signal for a FID is in picoamps (pa). Bleed levels are usually reported as the difference (DpA) in the FID signal at two temperatures. Column Installation & Conditioning Proper column installation is important to obtain the highest performance and lifetime from capillary GC columns. Prior to column installation it is important to check gas traps, gas flows, cylinder pressures, liners or any other area that requires periodic adjustment, cleaning (deactivation) or replacement. Using a septum to hold the nut and ferrule in place install the column into the injector as shown opposite. It is vital that the correct (o.d.) ferrule is used and that the column cut is even (not jagged) and at 90 o to the column to ensure good peak shape. Follow instrument manufacturers recommendations for the installation distance into the inlet failure to comply will result in broad peaks and poor area reproducibility. Once installed in the injector the carrier gas flow should be established. It is vital that the oven is NOT on at this point and carrier is allowed to flow through the column at ambient temperature for at least 10 minutes. Column flow can be confirmed by observing a steady stream of bubbles when submerging the column end into hexane (or alternative light solvent). Install the column into the detector following the same precautions as for the inlet and manufacturers guidelines. Condition the column (after at least 10 minutes of carrier flow at ambient temperature), by increasing the oven temperature to the isothermal temperature limit or o C above the upper temperature specified in the GC method (don t exceed the column isothermal maximum!). The length of time and specific conditioning requirements will depend on phase type, column length and film thickness. Crawford Scientific 41

43 Table 12. Ferrule selection Column length (m) Column I.D. (mm) Column Cutting Fused silica columns: Using a ceramic wafer, diamond tipped pencil or sapphire cutting tool, lightly scribe the column 2-4 cm from the end. Do not try to cut through the tubing, only scratch the polyimide coating. Grasp the column on each side of the scribe. Simultaneously pull outward and bend the tubing away from the scribe point. The column should easily and cleanly break at the scribe point. Inspect the cut edge with a 10-20X magnifier. The cut end should be at a 90 o angle relative to the tubing wall. There should be no burrs or large, jagged areas. If necessary, re-cut the column until a proper end is obtained. Crawford Scientific 42

44 Column Installation Only secure the column (using the nut) enough for gas tight operation at the upper column temperature. This is usually achieved by tightening the column with a wrench ¼ turn past finger tight. This will ensure good gas tightness and good performance from the ferrule. Preparing the column Crawford Scientific 43

45 Importance of column cuts i Conditioning Heat the column at its isothermal temperature limit. Alternatively, a temperature o C above the highest operating temperature (without exceeding the column s temperature limit) can be used. Using a temperature program is not necessary. Plot the baseline after the column has reached the conditioning temperature. Upon reaching the conditioning temperature, the baseline will continue to rise for 5-30 minutes then drop for another minutes. A flat baseline should be obtained 1-3 hours after reaching the conditioning temperature. If the baseline does not stabilize after 2-3 hours or does not remain constant, stop the conditioning process. Usually a leak in the carrier gas line or injector area, or system contamination, is responsible for unstable baseline. Crawford Scientific 44

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