Basic Gas Chromatography Prepared by Mina S Buenafe

Basic Gas Chromatography Prepared by: Mina S. Buenafe

Gas Chromatography • Chromatography – A Very Brief History • Definitions / Terminologies in GC • Instrumentation Overview • System Modules • Mobile Phase (Carrier Gas) • Inlets • Stationary Phase(s) • Columns (Packed and Capillary) • Detector(s) • Troubleshooting

Chromatography – A (Very) Brief History IN THE EARLY 1900’S M. Tswett published his work on separation of plant pigments. He coined the term chromatography (literally translated as color writing) and scientifically described the process – earning him the title “Father of Chromatography” W. Ramsey published his work on separation of mixture of gases and vapors on adsorbents like charcoal. IN THE EARLY 1940’s A. Martin and R. Synge first suggested the possibilities of gas chromatography in a paper published in Biochem. J. , v. 35, 1358, (1941). Martin won a Nobel Prize for his work in Partition chromatography. IN THE EARLY 1950’s A. Martin and A. James published the epic paper describing the first gas chromatograph

Definition of Terms Chromatography: A physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction “Official” IUPAC definition

Definition of Terms Chromatogram It is the output signal from the detector of the instrument.

Definition of Terms Distribution Constant (KC) It is the tendency of a given component to be attracted to the stationary phase. This can be expressed in chemical terms as an equilibrium constant. Also called the partition coefficient (KP) or the distribution coefficient (KD) KC = [A]S/[A]M Mathematically, it is defined as the concentration of solute A in the stationary phase divided by its concentration in the mobile phase.

Definition of Terms The attraction to the stationary phase can also be classified according to the type of sorption by the solute. Adsorption: sorption on the surface of the stationary phase Absorption: sorption into the bulk of the stationary phase (usually called ‘partition’ by chromatographers)

Definition of Terms Retention Volume (VR) It is usually defined as the distance between the point of injection to the peak maximum. It is the volume of the carrier gas necessary to elute the solute of interest. Mathematically: VR = FC x t. R Where FC is the constant flow rate t. R is the retention time

Definition of Terms Phase Ratio (b) For packed columns: b = Mobile Phase Volume Stationary Phase Volume For capillary columns: b = rc/2 df Where rc is the radius of the column df is the thickness of the film

Definition of Terms Retention Factor (k) It is the ratio of the amount of the solute (NOT concentration) in the stationary phase to the amount in the mobile phase. It is also called capacity factor (k’), capacity ratio, or partition ratio Mathematically: k = (WA)S/(WA)M = KC/b Also k = (t. R - t 0) = time in stationary phase t 0 time in carrier gas k is temperature and flow dependent. Best separations occur when k is between 5 and 7

Definition of Terms Theoretical Plates (N) This is the most common measure of column efficiency in chromatography N = 16(t. R/Wb)2 = 5. 54(t. R/ Wh)2 Where Wb is the peak width at the base Wh is the peak width at half-height

Definition of Terms Height Equivalent to a Theoretical Plate (H) This is a related parameter that also defines column efficiency. Also identified as HETP Mathematically: H = L/N Where L is the Column Length (An efficient column will have a large N and a small H)

Definition of Terms Separation Factor (a) It is a measure of relative distribution constants. Also known as selectivity and/or solvent efficiency. Mathematically: a = k 2/k 1 = (KC)2/(KC)1 It is dependent on: n Chemical composition of the phase n Partitioning between the two phases

Definition of Terms Resolution (Rs) It is the degree to which adjacent peaks are separated. Mathematically: Rs = (t. R)B – (t. R)A [(Wb)B + (Wb)A]/2 — Also Rs = L/H x k/(k+1) x a-1/a

Instrumentation Overview Schematic of a Typical Gas Chromatograph

System Modules Carrier Gas Main purpose: carries the sample through the column Secondary purpose: provides a suitable matrix for the detector to measure the sample component. Detector Carrier Gas Thermal Conductivity Helium Flame Ionization Helium or Nitrogen Electron Capture Very dry Nitrogen or Argon, 5% Methane Carrier gases should be of high purity (minimum of 99. 995%). • Oxygen & water impurities can chemically attack the liquid phase of the column and destroy it. • Trace water content can desorb other column contaminants and produce high detector background or ‘ghost peaks’. • Trace hydrocarbon contents can cause high detector background with FID’s and limit detectability.

System Modules Carrier Gas Flow Measurements and Control: • Essential for column efficiency and qualitative analysis (e. g. reproducibility of retention times) Average linear flow velocity (ū) in OT columns: ū = L/tm where L is column length in cm tm is the retention time of an unretained peak (e. g. methane) in sec To convert linear flow velocity to flow rate (Fc) in m. L/min: Fc = ū x Pr 2 x 60 sec/min

System Modules Carrier Gas Effect of mobile phase (carrier gas) density on column efficiency. Van Deemter plots for the 3 common carrier gases for a column of capacity factor k’ = 7. 90. The low density gases (H 2 & He) have optimum efficiency at slightly higher flow rates than N 2. The much lower slopes of H 2 and He curves allow them to be used at higher flow rates (compared to N 2) with very little loss of separation efficiency.

System Modules Inlets are the points of sample introduction Ideal Sample Inlets for Column Type: Packed Columns Flash Vaporizer Capillary Columns Split On-Column Splitless On-Column

System Modules Inlets Split Injector The oldest, simplest, and easiest injection technique. Cross section of a typical split injector Advantages to Split Injection: Disadvantages: • High resolution separations • Trace analysis is limited • Neat samples can be introduced. • Process sometimes discriminates between high molecular weight solute so that the sample entering the column is not representative of the sample injected. • Dirty samples can be introduced by putting a deactivated glass wool plug in the liner to trap non-volatile components

System Modules Inlets Splitless Injector Samples have to be diluted in a volatile solvent and 1 -5 m. L is injected in the heated injection port. Septum purge is essential in splitless injections. Cross section of a typical splitless injector Advantages to Splitless Injection: Disadvantages: • Improved sensitivity over a split injector • Time consuming • Initial temperature and time of opening the split valve needs to be optimized. • Not well suited for volatile compounds (boiling points of peaks of interest have to be about 30 o. C higher than solvent.

System Modules Inlets Other Types of Inlets: • Direct Injection: involves injecting a small sample into a glass liner where vapors are carried directly into the column. • On-Column Injection: inserting the precisely aligned needle into the capillary column and making injections inside the column. • Flash Vaporization: involves heating the injection port to a temperature well above the boiling point to ensure rapid volatilization • Static Headspace: concentrates the vapors over a solid or liquid sample (best for residual solvent analysis)

System Modules Stationary Phase Sub-classification of GC Techniques • GSC: gas solid chromatography stationary phase is solid • GLC: gas liquid chromatography stationary phase is liquid

System Modules Stationary Phase Gas Solid Chromatography (GSC) • Solids used are traditionally run in packed columns • These solids should have small and uniform particle sizes (e. g. 80/100 mesh range) Some Common GC Adsorbents Commercial/Trade Names Silica Gel Chromasil®, Porasil® Activated Alumina F-1, Unibeads-A® Zeolite Molecular Sieves MS 5 A, MS 13 X Carbon Molecular Sieves Carbopack®, Carbotrap®, Carbograph®, Graphpac® Porous Polymers Porapak®, Haye. Sep®, Chromosorb® Tenax Polymers Texan TA®, Tenax GR® • Some of these solids have been coated on the inside walls of capillary columns and are called “Support Coated Open Tubular” or SCOT columns.

System Modules Stationary Phase One major application of Packed Column GSC is in Gas Analysis. Reasons: • Adsorbents provide high surface areas for maximum interaction with gases that may be difficult to retain on liquid stationary phase. • Large samples can be accommodated, providing lower absolute detection limits. • Some packed column GC’s can be configured to run below ambient temperature which will also increase the retention of the gaseous solutes. • Unique combinations of multiple columns and/or valving make it possible to optimize analysis of a particular sample. Packed Columns also provide the flexibility of allowing mixed packings for special applications (e. g. 5% Fluorcol on Carbopack B® for analysis of Freons)

System Modules Stationary Phase Gas Liquid Chromatography (GLC) To use liquid as stationary phase, techniques were applied to hold the liquid in a column. • For packed columns: liquid is coated onto a solid support, chosen for its high surface area and inertness. The coated support is then dry-packed into a column as tightly as possible. • For capillary or open tubular (OT) columns: liquid is coated on the inside of the capillary. To make it adhere better, the liquid phase is often extensively crosslinked and sometimes chemically bonded to the fused silica surface. Schematic representation of (a) packed column and (b) capillary column

System Modules Stationary Phase Gas Liquid Chromatography (GLC) Requirements for the stationary liquid phase: • Low vapor pressure • Thermal stability • (if possible) Low viscosity (for fast mass transfer) • Should interact with the components of the sample to be analyzed (“Like dissolves like”)

System Modules Stationary Phase Gas Liquid Chromatography (GLC) Types of Capillary Columns (OT) WCOT: Wall-coated open tubular column (provides the highest resolution of all OT’s – i. d. ’s range from 0. 1 mm to 0. 53 mm and film thickness from 0. 1 – 5. 0 m) PLOT: Porous layer open tubular column (less than 5% of all GC use these days) SCOT: Surface-coated open tubular column (no longer available in fused silica)

System Modules Detectors The part of the system that ‘senses’ the effluents from the column and provides a record of the analysis in the form of a chromatogram. The signals are proportional to the quantity of each analyte.

System Modules Detectors FID: Flame Ionization Detector The most common GC detector used. The column effluent is burned in a small oxy-hydrogen flame producing some ions in the process. These ions are collected and form a small current that becomes the signals. When no sample is being burned, there should be little ionization, the small current is produced from impurities from the hydrogen and air supplies. Hydrogen flow rate is commonly set to 40 – 45 m. L/min, Air, 350 - 450 m. L/min, and for OT columns (with flows of about 1 m. L/min), Make-Up gases is added to carrier gas (to make up the flow to 30 m. L/min)

System Modules Detectors TCD: Thermal Conductivity Detector This is a differential detector that measures thermal conductivity of the analyte in the carrier gas compared to thermal conductivity of the pure gas. At least two cavities are required. These cavities are drilled into a metal block and each contain a hot wire or filament. The filaments are incorporated into a Wheatstone Bridge Circuit (for resistance measurements). The choice of carrier gas will depend on thermal conductivity of the analyte (H 2 and He have highest TC’s, N 2 gives rise to unusual peak shapes)

Detectors System Modules NPD: Nitrogen Phosphorus Detector A bead of Rb or Cs is electrically heated when flame ionization occurs. The detector shows enhanced detectability for nitrogen-, phosphorus-, or halogen- containing samples.

System Modules Detectors MSD: Mass Spectrometric Detector Analyte molecules are first ionized in order to be attracted or repelled by the proper magnetic or electrical fields.

Troubleshooting Common GC Problems: • Retention Time Problems • Resolution Problems • Baseline Problems • Peak Problems

Retention Time Problems Retention Time Shift Possible Cause Solution Comments Change in carrier velocity Check the carrier gas velocity All peaks will shift in the same direction by approximately the same amount Change in column temperature Check the column temperature Not all peaks will shift by the same amount Change in column dimensions Verify the column identity Large change in Try a different sample compound concentration May also affect adjacent peaks. Sample overloading is corrected with an increased split ratio, sample dilution, or decreased injection volume. Leak in the injector or column connection Usually accompanied by peak size change. Leak-check the injector and column installation

Retention Time Problems Retention Time Shift Possible Cause Solution Comments Blockage in a gas line Clean or replace the plugged line More common for the split line; also check flowcontrollers and solenoids. Septum leak Replace the septum Check for needle barb. Sample solvent incompatibility Change solvent. Use a retention gap. For splitless injector. Contamination Trim the column. Remove ½ - 1 meter from the front of the column. Only for bonded and crosslinked phase. Solvent-rinse the column.

Resolution Problems Loss of Resolution Decrease in separation Possible Cause Solution Comments Different column temperature Check column temperature Differences in other peaks will be visible Different column dimensions or phase Verify column identity Differences in other peaks will be visible Co-elution with another peak Change the column temperature Decrease column temperature and check the appearance of a peak shoulder or tail. Column contamination – resulting in a change in column selectivity Trim the column Remove ½ - 1 meter from the front of the column. Only for bonded and cross -linked phase. Solvent-rise the column

Resolution Problems Loss of Resolution Increase in peak width Possible Cause Solution Comments Change in carrier gas velocity Check carrier gas velocity A change in retention time also occurs Column contamination Trim the column Remove ½ - 1 meter from the front of the column. Only for bonded and crosslinked phase. Solvent-rise the column Inlet liner contamination Clean or replace liner Change in the injector Check the injector settings Typical areas: split ratio, liner, temperature, injection volume Change in the sample concentration Try a different sample concentration Peak width increases at higher concentration Improper solvent effect lack of focusing Lower oven temperature. Choose different solvent for better solvent/sample/phase polarity match. Use a retention gap For splitless injection.

Baseline Problems • Excessive Column Bleed Possible Cause Thermal damage to the column Oxygen damage to column Chemical phase damage to column Solution Comments Remove column from detector and bake-out overnight, reinstall and condition as usual Use GC maximum temperature function Columns damaged by oxygen will usually need to be replaced although an overnight bake-out may be attempted Perform periodic leak checks. Change septa regularly. Use high quality carrier gases. Install and maintain oxygen traps Remove ½ to 5 meters from the front of the column Perform sample prep to remove inorganic acids and bases from the sample. Install guard column and trim frequently. If acids or bases must be used, choose HCl or NH 4 OH, or an organic alternative.

Baseline Problems • Erratic Baseline (drift, wander) Possible Cause Inlet contamination Solution Clean the injector Comments Try a condensation test; gas lines may also need cleaning. Take steps to prevent sample backflash (reduce injection volume, lower inlet temperature, use larger volume liner Column contamination Bake-out column. Solvent-rinse the column Limit bake-out to 1 – 2 hours Only for bonded and cross-linked phases. Check for inlet contamination Incompletelyconditioned column Fully condition the column More critical for trace analysis Un-equilibrated detector Allow the detector to stabilize Some detectors may require up to 24 hours to fully stabilize Change in carrier gas flow-rate during the temperature program Normal in many cases MS, TCD, and ECD respond to carrier gas flow rate changes

Baseline Problems • Erratic Baseline (drift, wander) Possible Cause Solution Comments Contaminated gases Use appropriate purifier to remove contaminants More of a problem for detector gases. Column and inlet liner misaligned Check installation of column end and inlet liner, adjust if necessary Causes a baseline change after a large peak Large leak at the septum Replace septum during injection and for a Use smaller diameter needle short time thereafter Sample decomposing Causes a baseline change after a large peak. Common with large diameter needles. Remove inlet liner and check Causes a baseline rise cleanliness. before and after a peak. Use new, deactivated liner or replace glass wool and packing.

Baseline Problems ·Noisy Baseline Possible Cause Solution Comments Inlet contamination Clean the injector, replace liner, gold seal Try a condensation test; gas lines may also need cleaning. Column contamination Limit bake-out to 1 – 2 hours Bake-out the column. Only for bonded and cross. Solvent-rinse the column. linked phases. Check for inlet contamination Detector contamination Clean the detector. Contaminated or low quality gases Replace spent gas purifier. Use purifiers to remove contaminants. Use better grade gases. Column inserted too far into the detector Reinstall the column. Usually the noise increases over time and not suddenly. More of a problem for detector gases. Consult the GC manual for the proper installation distance.

·Noisy Baseline Problems Baseline Possible cause Solution Comments Incorrect detector gas flow rates Adjust the flow rates to the recommended values. Consult the GC manual for appropriate flow rates Leak when using an MS, ECD, or TCD Find and eliminate the leak. Usually at the column fittings or injector. Old detector filament, lamp, or electron multiplier, NPD head Replace appropriate part. Septum degradation Replace septum. For high temperature applications, use appropriate septum

Baseline Problems ·Ghost Peaks Possible Cause Contaminants introduced with sample Solution Sample or solvent clean-up Comments Contaminants in sample process or solvent. Clean the injector, replace liner, gold seal, and septum Try a condensation test; gas lines may also need cleaning. Take steps to prevent sample backflash (reduce injection volume, lower inlet temperature, use larger volume liner) Septum bleed Replace septum. Use a high quality septum appropriate for the inlet temperature. Contamination of sample prior to introduction to the GC Check sample handling steps for potential contamination sources: sample clean-up, handling, transfer, and storage Semi-volatile contamination (peak widths will be broader than sample peaks with similar retention Bake out column, Solvent-rinse the column. Check for contamination in the inlet, carrier gas, or carrier gas lines. Inlet contamination Limit bake-out to 1 – 2 hours. Only for bonded and cross-linked phases.

Peak Problems ·Fronting Peaks Possible cause Solution Comments Reduce mass amount of the analyze to the column. Decrease injection volume, dilute sample, increase split ratio Most common cause for fronting peaks. Reinstall the column in the injector. Consult the GC manual for the proper installation distance Injection technique. Change technique. Usually related to erratic plunger depression or having sample in the syringe needle. Use an autosampler. Compound very soluble in injection solvent Change solvent. Using a retention gap may help. Column overload Improper column installation. Mixed sample solvent Change sample solvent. Worse for solvents with large differences in polarity or boiling points.

Peak Problems ·Tailing Peaks Possible cause Solution Comments Severe column contamination Trim the column. Solvent rinse the column Remove ½ - 1 meter from the front end of the column. Only for bonded or cross-linked phase. Check for inlet contamination. Tailing will sometimes increase with compound retention. Active column Cut off 1 -meter from the front end of the column. Replace column. Only affects active compounds. Usually produces tailing that increases with retention. Improper column installation, leak, or column end poorly cut Re-cut and reinstall the column into the inlet. Replace ferrule. Confirm installation is leak-free Make a clean square cut with a reliable cutting tool. Consult GC manual for the proper installation distance. More tailing for early eluting peaks. Contaminated or active liner or gold seal Use new, deactivated liner. Clean or replace gold seal. Only affects active compounds.

Peak Problems ·Tailing Peaks Possible cause Solution Solid particles in liner Clean or replace liner. Needle hitting and breaking packing in inlet liner Partially remove packing from liner or use without packing. Solvent/column not compatible Use a different solvent. Use a retention gap. Comments More tailing for the early eluting peaks or those closest to the solvent front. 3 – 5 meter retention gap is sufficient. Split ratio too low Increase split ratio. Flow from split vent should be 20 m. L/min Solvent effect violations for splitless or on-column injections Decrease the initial column temperature to 10 - 25°C below solvent boiling point Peak tailing decreases with retention. Poor injection technique Change technique Usually related to erratic plunger depression or having sample in the syringe needle. Use an autosampler.

Peak Problems ·Tailing Peaks Possible cause Solution Comments Inlet temperature too high Decrease inlet temperature by 50°C Tailing generally worse for early eluters. Inlet temperature too low Increase inlet temperature by 50°C Tailing usually increases with retention Dead volume in system Reduce dead volume. Transfer line connections, fused silica unions, etc. Peak tailing decreases with retention. Cold spots (condensation) Eliminate cold spots. Commonly at transfer lines. Tailing usually increases with retention Overloading of PLOT columns Reduce the amount injected onto column.

Peak Problems ·Split Peaks Possible cause Solution Comments Reinstall the column in the injector Consult the GC manual for the proper installation distance Change technique. Usually related to erratic plunger depression or having sample in the syringe needle. Use an autosampler. Mixed sample solvent Change sample solvent. Worse for solvents with large differences in polarity or boiling points Poor sample focusing Use a retention gap Solvent/column not compatible Use a different solvent. Use a retention gap. Column installation Injection technique For splitless, on-column, and PTV injectors

Peak Problems ·Split Peaks Possible cause Sample degradation in injector (only some peaks show splitting) Solution Reduce inlet temperature. Derivatize sample to make compounds thermally stable. Change to an on-column injector. Comments Peak broadening or tailing may occur if the temperature is too low. Requires an on-column injector Severe detector overload Reduce the amount of sample on-column. May only affect some peaks.

Peak Problems ·Changes in Peak Size Possible cause Solution Comments Change in detector response Check gas flow, temperature and settings. Check background level or noise All peaks may not be equally affected May be caused by the system contamination, not the detector. Change in the split ratio Check the split ratio All peaks may not be equally affected. Change in the purge Check the purge activation time For splitless injectors Change in injection volume Check injection technique Injection volumes are not linear. Change in injector discrimination Maintain the same injector Most severe for spit parameters: flows, injections. All peaks may temperatures, liners, etc. not be equally affected

Peak Problems ·Changes in Peak Size Possible cause Solution Comments Change in sample concentration Check and verify sample concentration May be caused by degradation, evaporation, or variances in sample temperature or p. H Leak in the syringe Use a different syringe Sample leas past the plunger or around the needle; leaks are often not readily visible Column contamination Trim the column Remove ½ - 1 meter from the front of the column. Only for bonded and crosslinked phases Solvent-rinse the column Column activity Trim or replace the column Only affects active compounds

• Peak Problems ·Changes in Peak Size Possible Cause Solution Comments Co-elution Change column temperature or stationary phase Decrease column temperature, and check for the appearance of peak shoulder or tail Sample backflash Inject less, use larger liner, or reduce the inlet temperature Less solvent and higher flow rates are most helpful Decomposition from inlet contamination Clean the inlet, replace the liner, replace the gold seal Only use deactivated liners and glass wool in the inlet. Loss of sample prior Check sample handling, to introduction into sample preparation, the GC transfer and storage

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