Presentation on theme: "Basic Gas Chromatography"— Presentation transcript:
1 Basic Gas Chromatography Prepared by: Mina S. Buenafe
2 Gas Chromatography Chromatography – A Very Brief History Definitions / Terminologies in GCInstrumentation OverviewSystem ModulesMobile Phase (Carrier Gas)InletsStationary Phase(s)Columns (Packed and Capillary)Detector(s)Troubleshooting
3 Chromatography – A (Very) Brief History IN THE EARLY 1900’SM. 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’sA. 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’sA. Martin and A. James published the epic paper describing the first gas chromatograph
4 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
5 Definition of Terms Chromatogram It is the output signal from the detector of the instrument.
6 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]MMathematically, it is defined as the concentration of solute A in the stationary phase divided by its concentration in the mobile phase.
7 Definition of TermsThe 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 phaseAbsorption: sorption into the bulk of the stationary phase (usually called ‘partition’ by chromatographers)
8 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 tRWhere FC is the constant flow ratetR is the retention time
9 Definition of Terms Phase Ratio (b) For packed columns: b = Mobile Phase VolumeStationary Phase VolumeFor capillary columns:b = rc/2dfWhere rc is the radius of the columndf is the thickness of the film
10 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 ratioMathematically:k = (WA)S/(WA)M = KC/bAlso k = (tR - t0) = time in stationary phaset time in carrier gask is temperature and flow dependent. Best separations occur when k is between 5 and 7
11 Definition of Terms Theoretical Plates (N) This is the most common measure of column efficiency in chromatographyN = 16(tR/Wb)2 = 5.54(tR/ Wh)2Where Wb is the peak width at the baseWh is the peak width at half-height
12 Definition of Terms Height Equivalent to a Theoretical Plate (H) This is a related parameter that also defines column efficiency. Also identified as HETPMathematically:H = L/NWhere L is the Column Length(An efficient column will have a large N and a small H)
13 Definition of Terms Separation Factor (a) It is a measure of relative distribution constants. Also known as selectivity and/or solvent efficiency.Mathematically: a = k2/k1 = (KC)2/(KC)1It is dependent on:Chemical composition of the phasePartitioning between the two phases
14 Definition of Terms Resolution (Rs) It is the degree to which adjacent peaks are separated.Mathematically:Rs = (tR)B – (tR)A[(Wb)B + (Wb)A]/2Also Rs =—L/H x k/(k+1) x a-1/a
15 Instrumentation Overview Schematic of a Typical Gas Chromatograph
16 Very dry Nitrogen or Argon, 5% Methane System ModulesCarrier GasMain purpose: carries the sample through the columnSecondary purpose: provides a suitable matrix for the detector to measure the sample component.DetectorCarrier GasThermal ConductivityHeliumFlame IonizationHelium or NitrogenElectron CaptureVery dry Nitrogen or Argon, 5% MethaneCarrier gases should be of high purity (minimum of %).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.
17 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/tmwhere L is column length in cmtm is the retention time of an unretained peak (e.g. methane) in secTo convert linear flow velocity to flow rate (Fc) in mL/min:Fc = ū x Pr2 x 60sec/min
18 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’ = The low density gases (H2 & He) have optimum efficiency at slightly higher flow rates than N2. The much lower slopes of H2 and He curves allow them to be used at higher flow rates (compared to N2) with very little loss of separation efficiency.
19 System Modules Inlets Inlets are the points of sample introduction Ideal Sample Inlets for Column Type:Packed ColumnsCapillary ColumnsFlash VaporizerSplitOn-ColumnSplitless
20 Cross section of a typical split injector System ModulesInletsSplit InjectorThe oldest, simplest, and easiest injection technique.Cross section of a typical split injectorAdvantages to Split Injection:High resolution separationsNeat samples can be introduced.Dirty samples can be introduced by putting a deactivated glass wool plug in the liner to trap non-volatile componentsDisadvantages:Trace analysis is limitedProcess sometimes discriminates between high molecular weight solutes so that the sample entering the column is not representative of the sample injected.
21 Cross section of a typical splitless injector System ModulesInletsSplitless InjectorSamples have to be diluted in a volatile solvent and 1-5mL is injected in the heated injection port. Septum purge is essential in splitless injections.Cross section of a typical splitless injectorAdvantages to Splitless Injection:Improved sensitivity over a split injectorDisadvantages:Time consumingInitial 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 30oC higher than solvent.
22 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 volatilizationStatic Headspace: concentrates the vapors over a solid or liquid sample (best for residual solvent analysis)
23 System Modules Stationary Phase Sub-classification of GC Techniques GSC: gas solid chromatography - stationary phase is solidGLC: gas liquid chromatography -stationary phase is liquid
24 System Modules Stationary Phase Gas Solid Chromatography (GSC) Solids used are traditionally run in packed columnsThese solids should have small and uniform particle sizes (e.g. 80/100 mesh range)Some Common GC AdsorbentsCommercial/Trade NamesSilica GelChromasil®, Porasil®Activated AluminaAlumina F-1, Unibeads-A®Zeolite Molecular SievesMS 5A, MS 13XCarbon Molecular SievesCarbopack®, Carbotrap®, Carbograph®, Graphpac®Porous PolymersPorapak®, HayeSep®, Chromosorb®Tenax PolymersTexan 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.
25 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)
26 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 cross-linked and sometimes chemically bonded to the fused silica surface.Schematic representation of (a) packed column and (b) capillary column
27 System Modules Stationary Phase Gas Liquid Chromatography (GLC) Requirements for the stationary liquid phase:Low vapor pressureThermal stability(if possible) Low viscosity (for fast mass transfer)Should interact with the components of the sample to be analyzed (“Like dissolves like”)
28 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 0.1mm to 0.53mm and film thickness from 0.1 – 5.0m)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)
29 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.
30 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 from the hydrogen and air supplies.Hydrogen flow rate is commonly set to 40 – 45mL/min, Air, mL/min, and for OT columns (with flows of about 1 mL/min), Make-Up gases is added to carrier gas (to make up the flow to 30mL/min)
31 System Modules Detectors TCD: Thermal Conductivity Detector This is a differential detector that measures the thermal conductivity of the analyte in the carrier gas compared to the 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 the thermal conductivity of the analyte (H2 and He have highest TC’s, N2 gives rise to unusual peak shapes)
32 System Modules Detectors 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.
33 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.
34 Troubleshooting Common GC Problems: Retention Time Problems Resolution ProblemsBaseline ProblemsPeak Problems
35 Retention Time Problems Retention Time ShiftPossible CauseSolutionCommentsChange in carrier velocityCheck the carrier gas velocityAll peaks will shift in the same direction by approximately the same amountChange in column temperatureCheck the column temperatureNot all peaks will shift by the same amountChange in column dimensionsVerify the column identityLarge change in compound concentrationTry a different sample concentrationMay 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 connectionLeak-check the injector and column installationUsually accompanied by peak size change.
36 Retention Time Problems Retention Time ShiftPossible CauseSolutionCommentsBlockage in a gas lineClean or replace the plugged lineMore common for the split line; also check flow-controllers and solenoids.Septum leakReplace the septumCheck for needle barb.Sample solvent incompatibilityChange solvent. Use a retention gap.For splitless injector.ContaminationTrim the column.Solvent-rinse the column.Remove ½ - 1 meter from the front of the column.Only for bonded and cross-linked phase.
37 Resolution Problems Loss of Resolution Decrease in separation Possible CauseSolutionCommentsDifferent column temperatureCheck column temperatureDifferences in other peaks will be visibleDifferent column dimensions or phaseVerify column identityCo-elution with another peakChange the column temperatureDecrease column temperature and check the appearance of a peak shoulder or tail.Column contamination – resulting in a change in column selectivityTrim the columnSolvent-rise the columnRemove ½ - 1 meter from the front of the column.Only for bonded and cross-linked phase.
38 Resolution Problems Loss of Resolution Increase in peak width Possible CauseSolutionCommentsChange in carrier gas velocityCheck carrier gas velocityA change in retention time also occursColumn contaminationTrim the columnSolvent-rise the columnRemove ½ - 1 meter from the front of the column.Only for bonded and cross-linked phase.Inlet liner contaminationClean or replace linerChange in the injectorCheck the injector settingsTypical areas: split ratio, liner, temperature, injection volumeChange in the sample concentrationTry a different sample concentrationPeak width increases at higher concentrationImproper solvent effect lack of focusingLower oven temperature.Choose different solvent for better solvent/sample/phase polarity match.Use a retention gapFor splitless injection.
39 Baseline Problems Excessive Column Bleed Possible Cause Solution CommentsThermal damage to the columnRemove column from detector and bake-out overnight, reinstall and condition as usualUse GC maximum temperature functionOxygen damage to columnColumns damaged by oxygen will usually need to be replaced although an overnight bake-out may be attemptedPerform periodic leak checks. Change septa regularly. Use high quality carrier gases. Install and maintain oxygen trapsChemical phase damage to columnRemove ½ to 5 meters from the front of the columnPerform 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 NH4OH, or an organic alternative.
40 Baseline Problems Erratic Baseline (drift, wander) Possible Cause SolutionCommentsInlet contaminationClean the injectorTry a condensation test; gas lines may also need cleaning. Take steps to prevent sample backflash (reduce injection volume, lower inlet temperature, use larger volume linerColumn contaminationBake-out column.Solvent-rinse the columnLimit bake-out to 1 – 2 hoursOnly for bonded and cross-linked phases.Check for inlet contaminationIncompletely-conditioned columnFully condition the columnMore critical for trace analysisUn-equilibrated detectorAllow the detector to stabilizeSome detectors may require up to 24 hours to fully stabilizeChange in carrier gas flow-rate during the temperature programNormal in many casesMS, TCD, and ECD respond to carrier gas flow rate changes
41 Baseline Problems Erratic Baseline (drift, wander) Possible Cause SolutionCommentsContaminated gasesUse appropriate purifier to remove contaminantsMore of a problem for detector gases.Column and inlet liner misalignedCheck installation of column end and inlet liner, adjust if necessaryCauses a baseline change after a large peakLarge leak at the septum during injection and for a short time thereafterReplace septumUse smaller diameter needleCauses a baseline change after a large peak.Common with large diameter needles.Sample decomposingRemove inlet liner and check cleanliness.Use new, deactivated liner or replace glass wool and packing.Causes a baseline rise before and after a peak.
42 Baseline Problems ·Noisy Baseline Possible Cause Solution Comments Inlet contaminationClean the injector, replace liner, gold sealTry a condensation test; gas lines may also need cleaning.Column contaminationBake-out the column.Solvent-rinse the column.Limit bake-out to 1 – 2 hoursOnly for bonded and cross-linked phases.Check for inlet contaminationDetector contaminationClean the detector.Usually the noise increases over time and not suddenly.Contaminated or low quality gasesReplace spent gas purifier.Use purifiers to remove contaminants.Use better grade gases.More of a problem for detector gases.Column inserted too far into the detectorReinstall the column.Consult the GC manual for the proper installation distance.
43 Baseline Problems ·Noisy Baseline Possible cause Solution Comments Incorrect detector gas flow ratesAdjust the flow rates to the recommended values.Consult the GC manual for appropriate flow ratesLeak when using an MS, ECD, or TCDFind and eliminate the leak.Usually at the column fittings or injector.Old detector filament, lamp, or electron multiplier, NPD headReplace appropriate part.Septum degradationReplace septum.For high temperature applications, use appropriate septum
44 Baseline Problems ·Ghost Peaks Possible Cause Solution Comments Contaminants introduced with sampleSample or solvent clean-upContaminants in sample process or solvent.Inlet contaminationClean the injector, replace liner, gold seal, and septumTry 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 bleedReplace septum.Use a high quality septum appropriate for the inlet temperature.Contamination of sample prior to introduction to the GCCheck sample handling steps for potential contamination sources: sample clean-up, handling, transfer, and storageSemi-volatile contamination (peak widths will be broader than sample peaks with similar retentionBake out column,Solvent-rinse the column.Check for contamination in the inlet, carrier gas, or carrier gas lines.Limit bake-out to 1 – 2 hours.Only for bonded and cross-linked phases.
45 Peak Problems ·Fronting Peaks Possible cause Solution Comments Column overloadReduce mass amount of the analyze to the column. Decrease injection volume, dilute sample, increase split ratioMost common cause for fronting peaks.Improper column installation.Reinstall the column in the injector.Consult the GC manual for the proper installation distanceInjection technique.Change technique.Usually related to erratic plunger depression or having sample in the syringe needle.Use an autosampler.Compound very soluble in injection solventChange solvent. Using a retention gap may help.Mixed sample solventChange sample solvent.Worse for solvents with large differences in polarity or boiling points.
46 Peak Problems ·Tailing Peaks Possible cause Solution Comments Severe column contaminationTrim the column.Solvent rinse the columnRemove 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 columnCut 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 cutRe-cut and reinstall the column into the inlet.Replace ferrule.Confirm installation is leak-freeMake 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 sealUse new, deactivated liner.Clean or replace gold seal.Only affects active compounds.
47 Peak Problems ·Tailing Peaks Possible cause Solution Comments Solid particles in linerClean or replace liner.Needle hitting and breaking packing in inlet linerPartially remove packing from liner or use without packing.Solvent/column not compatibleUse a different solvent.Use a retention gap.More tailing for the early eluting peaks or those closest to the solvent front.3 – 5 meter retention gap is sufficient.Split ratio too lowIncrease split ratio.Flow from split vent should be 20mL/minSolvent effect violations for splitless or on-column injectionsDecrease the initial column temperature to °C below solvent boiling pointPeak tailing decreases with retention.Poor injection techniqueChange techniqueUsually related to erratic plunger depression or having sample in the syringe needle.Use an autosampler.
48 Peak Problems ·Tailing Peaks Possible cause Solution Comments Inlet temperature too highDecrease inlet temperature by 50°CTailing generally worse for early eluters.Inlet temperature too lowIncrease inlet temperature by 50°CTailing usually increases with retentionDead volume in systemReduce dead volume. Transfer line connections, fused silica unions, etc.Peak tailing decreases with retention.Cold spots (condensation)Eliminate cold spots. Commonly at transfer lines.Overloading of PLOT columnsReduce the amount injected onto column.
49 Peak Problems ·Split Peaks Possible cause Solution Comments Column installationReinstall the column in the injectorConsult the GC manual for the proper installation distanceInjection techniqueChange technique.Usually related to erratic plunger depression or having sample in the syringe needle.Use an autosampler.Mixed sample solventChange sample solvent.Worse for solvents with large differences in polarity or boiling pointsPoor sample focusingUse a retention gapFor splitless, on-column, and PTV injectorsSolvent/column not compatibleUse a different solvent.Use a retention gap.
50 Peak Problems ·Split Peaks Possible cause Solution Comments Sample degradation in injector (only some peaks show splitting)Reduce inlet temperature.Derivatize sample to make compounds thermally stable.Change to an on-column injector.Peak broadening or tailing may occur if the temperature is too low.Requires an on-column injectorSevere detector overloadReduce the amount of sample on-column.May only affect some peaks.
51 Peak Problems ·Changes in Peak Size Possible cause Solution Comments Change in detector responseCheck gas flow, temperature and settings.Check background level or noiseAll peaks may not be equally affectedMay be caused by the system contamination, not the detector.Change in the split ratioCheck the split ratioAll peaks may not be equally affected.Change in the purge activation timeCheck the purge activation timeFor splitless injectorsChange in injection volumeCheck injection techniqueInjection volumes are not linear.Change in injector discriminationMaintain the same injector parameters: flows, temperatures, liners, etc.Most severe for spit injections. All peaks may not be equally affected
52 Peak Problems ·Changes in Peak Size Possible cause Solution Comments Change in sample concentrationCheck and verify sample concentrationMay be caused by degradation, evaporation, or variances in sample temperature or pHLeak in the syringeUse a different syringeSample leas past the plunger or around the needle; leaks are often not readily visibleColumn contaminationTrim the columnSolvent-rinse the columnRemove ½ - 1 meter from the front of the column.Only for bonded and cross-linked phasesColumn activityTrim or replace the columnOnly affects active compounds
53 Peak Problems ·Changes in Peak Size Possible Cause Solution Comments Co-elutionChange column temperature or stationary phaseDecrease column temperature, and check for the appearance of peak shoulder or tailSample backflashInject less, use larger liner, or reduce the inlet temperatureLess solvent and higher flow rates are most helpfulDecomposition from inlet contaminationClean the inlet, replace the liner, replace the gold sealOnly use deactivated liners and glass wool in the inlet.Loss of sample prior to introduction into the GCCheck sample handling, sample preparation, transfer and storage