Trajan SGE GC Columns

Transcription

1 Trajan Scientific and Medical Trajan SGE GC Columns

2 Trajan Scientific and Medical Our focus is on developing and commercializing technologies that enable analytical systems to be more selective, sensitive and specific for biological, environmental or food related measurements especially those that can lead to portability, miniaturization and affordability. Trajan Analytical Trajan Life Trajan LEAP Automation Trajan Accelerator

3 Trajan Analytical We are a key partner in producing critical components and materials for analytical workflow and measurement. A global provider of engineered components and advanced technologies with a strong heritage in chromatography and mass spectrometry. Our aim is to design and deliver a broad portfolio of comprehensive analytical tools to improve laboratory performance and enhance confidence in results. This organization is built from the foundations of SGE Analytical Science.

4 Trajan Analytical GC and GCMS Business Unit Workflow solutions to optimize GC and GCMS system capabilities We are world leaders in creating solutions for GC and GCMS, through expertise in surface chemistry and glass forming. Custom stationary phase synthesis Chemical surface treatments Workflow optimization expertise Manufacture of precision bore glass cylinders Inert glass surface treatments Connectors for easy installation and low dead volume

5 Trajan Scientific and Medical GC Columns

6 Learning outcomes The features of a GC column The resolution equation Effects of changing GC column parameters Altering GC selectivity through temperature and phase chemistry GC column modes of interaction GC column troubleshooting

7 Features of a GC column Choice of Column dimensions For example: BPX5 30 m x 0.25 mm x 0.25 µm

8 The resolution equation Efficiency Retention Selectivity RR ss = NN 4 kk kk + 1 αα 1 αα

9 Column variables effect on resolution Variable Contribution to resolution 1 Column internal diameter N 2 Stationary phase loading N, k 3 Column length N 4 Carrier gas N 5 Temperature k 6 Stationary phase composition α

10 Column length RR ss = NN 4 kk αα 1 kk + 1 αα Resolution increases according to the square root of the column efficiency. Doubling column length increases resolution by ~ 40% m (0.25 mm / 0.25 μm) 1.4 ml/min helium 15 m (0.25 mm / 0.25 μm) 1.4 ml/min helium A longer column will provide greater resolution than a shorter column Shorter column increases speed time (min)

11 Column length GC columns typically range in length from 10 m up to 120 m The standard length for a GC column is 25 m or 30 m, this provides a high efficiency with relatively short analysis time Columns of m in length are typically used for faster analysis and chromatography of higher molecular weight substances Columns of 60 m and greater in length are used for very complex samples

12 Column length effects

13 Column internal diameter RR ss = NN 4 kk αα 1 kk + 1 αα Halving diameter doubles efficiency (increase resolution by ~ 40%) Golay Plot (helium carrier; 1 atm outlet) 30 m x 0.53 mm I.D. The smaller the column I.D. the: H (mm) m x 0.32 mm I.D. Greater the efficiency Better the resolution m x 0.25 mm I.D. 30 m x 0.15 mm I.D Average linear velocity (cm/s) Lower the sample handling capacity (may result in column overloading and poor resolution / peak shape)

14 Column internal diameter mm I.D.: for high resolution and short retention times with low carrier gas flows (Fast-GC) 0.25 mm I.D.: for analyses of complex mixtures with high resolution 0.32 mm I.D.: for routine analyses with short retention times, but increased capacity 0.53 mm I.D.: for rapid separations with inert surface and highest capacity

15 Column length and internal diameter Column I.D. Column length (m) (mm) HH mmmmmm,ttttttttt = jj GG dd cc kk + 11kk kk 2 Can estimate efficiency using: NN LL dd cc 30,000 / 0.25 = 120,000

16 Column length and internal diameter BPX5 30 m 0.25 mm I.D μm film thickness BPX5 7 m 0.10 mm I.D μm film thickness

17 Column film thickness RR ss = NN 4 kk αα 1 kk + 1 αα Thicker film = increased retention resulting in: Increased resolution of highly volatile compounds Decreased resolution for late eluting compounds Increased elution temperature and analyte capacity Greater inertness Thinner film = reduced retention resulting in: Sharper peaks Improved signal to noise ratios Reduced column bleed Increased maximum operating temperature Increased analyte interaction with the tubing wall Decreased analyte capacity

18 Column film thickness A film thickness of µm is standard which allows for injection of samples with wide volatility Thinner ( µm) films are used for compounds with high molecular weight Thick films (1-5 µm) are used to separate solvents, gases and very volatile substances Increasing film thickness decreases thermal stability, leading to higher bleed levels, limiting the maximum operating temperature of the column

19 Column film thickness ββ = dd cc 4dd ff β phase ratio of the column d c column diameter (μm) d f film thickness (μm) Column I.D Column I.D Film thickness, d f (μm) d c (mm) d c (μm)

20 Column film thickness and phase ratio Extremely volatile compounds should be analyzed on thick film columns to increase the time the compounds spend in the stationary phase, allowing them to separate. High molecular weight compounds must be analyzed on thinner film columns. This reduces the length of time the analytes stay in the column, and minimizes bleed at required higher elution temperatures.

21 Chromatographer s triangle of compromise SPEED CAPACITY EFFICIENCY

22 Altering selectivity in GC Temperature Stationary Phase Chemistry

23 Temperature ramp rate Speed of analysis increases with increasing GC oven temperature ramp rate Increase in retention factor, but at the expense of resolution If resolution is sufficient, then high temperature ramp rate can be used

24 Temperature ramp rate Time (s)

25 Temperature ramp rate

26 Developing a temperature program Isothermal operation The solubility of the analyte in mobile phase is lower at lower temperature Peaks broaden as retention time increases

27 Linear velocity of carrier gas Speed of analysis and resolution increases with increasing linear velocity Under isothermal conditions: If linear velocity deviates from optimum linear velocity (U opt ), you see relative peak broadening and loss of resolution Increase in flow rate increases peak capacity Highest peak capacity will always be observed for an isothermal separation usually too slow, broadening can impact detectability The effect of the increased carrier gas flow on the temperature gradient is to decrease the temperature gradient relative to the time the compounds stay on the column

28 Linear velocity of carrier gas

29 Temperature ramp rate vs linear velocity

30 Stationary phase selection Phase and temperature directly affect selectivity Use the principle like dissolves like Separate polar analytes using a more polar phase and vice versa The skill is knowing the degree of polarity required to avoid long retention times whilst still obtaining a satisfactory separation Separating compounds of intermediate polarity or mixed polarity and functionality requires knowledge of the retentivity and selectivity of each phase Fine tuning of phase chemistry may be required

31 GC columns modes of separation GC separations are not just based on boiling point Understanding of the interactions of the stationary phase can aid method development There are three different mechanisms of retention when using GC Utilization of different columns will alter the degree of retention of each of these three primary mechanisms: Dispersive interactions Dipole-dipole (and dipole-induced dipole) Hydrogen bonding These can be demonstrated using a series of test probes injected onto a range of columns

32 What column chemistries are available? Polysiloxane stationary phase base units: Typical functional groups:

33 Impact of stationary phase on elution order Peak Number Compound Log P Boiling Point ( C) 1 Toluene Decane Heptanol Phenol Dodecane Naphthalene

34 Impact of stationary phase on elution order

35 100% dimethyl polysiloxane Peak Number Compound Peak Color 1 Toluene Red 4 Phenol Blue 3 1-Heptanol Black 2 Decane Orange 6 Naphthalene Green 5 Dodecane Purple 1 4, Dispersive interactions only

36 5% phenyl polysiloxane Peak Number Compound Peak Color 1 Toluene Red 3 Heptanol Black 4 Phenol Blue 2 Decane Orange 5 Dodecane Purple 6 Naphthalene Green 1 4, Dispersive interactions Weak dipole induced dipole interactions ,

37 50% phenyl methylpolysiloxane Peak Number Compound Peak Color 1 Toluene Red 2 Decane Orange 3 Heptanol Black 4 Phenol Blue 5 Dodecane Purple 6 Naphthalene Green 1 4, Moderate dispersive interactions Strong dipole-induced dipole interactions

38 100% polyethylene glycol Peak Number Compound Peak Color 2 Decane Orange 1 Toluene Red 5 Dodecane Purple 3 Heptanol Black 6 Naphthalene Green 4 Phenol Blue 1 4, Weak dispersive interactions moderate dipole-dipole Strong hydrogen bonding

39 Stationary phase selectivity 11 Methanol Acetone , BP , BPX5 Compound (bp.) 1. Ethyl Acetate (77.1) 2. Benzene ( 80.1) 3. Butanol (117.6) 4. Toluene (110.6) 5. Ethyl Benzene (136.2) 6. m-xylene (139.1) 7. p-xylene (138.3) 8. o-xylene (144) 9. Ethyl Hexanoate (168) 10. Decane (174.1) 11. Dodecane (216.3) Acetone 1 Methanol 3, SolGel-WAX

40 Trajan Scientific and Medical GC Column Troubleshooting

41 Optimizing column performance Column conditioning Essential for good chromatography and prolonged column lifetimes Must be done with carrier gas flow on Temperature limits Upper : Isothermal and temperature program limits Lower : column will not function correctly Column installation Correct positioning in detector and injector Cutting Column storage Seal ends appropriately

42 Baseline drifting - general Possible causes: Accumulation of impurities in the column Carrier gas cylinder pressure too low to allow control Drifting carrier gas or combustion gas flows Remedy: Remove the end section of the column Check for impurities in the carrier gas Replace or Install appropriate gas filters Replace the carrier gas cylinder and increase the pressure Check the gas controllers

43 Baseline rising - drift Possible causes: Damaged column or one that has been exposed to O 2 may experience some phase decomposition Column bleed A poorly conditioned column Detector contamination Carrier gas contamination Total Ion Count 4.00E E E+000 Remedy: Condition or re-condition the column or change the column Trace and repair the leak Check the detector and clean it Time (min) Check for impurities in the gas source, replace or install appropriate gas filters

44 Column bleed Column bleed is the normal background signal caused by stationary phase degradation: All columns exhibit some degree of bleed Column bleed will increase with film thickness and column dimensions Check for bleed by running a blank trace: Baseline rise should start ~40 below the column s isothermal limit Before and after the rise the baseline is level No peaks are eluted The trace will vary if the temperature profile is varied

45 Noise Possible causes: Contaminated injector and / or column Defective detector The column may be inserted too far into the flame of an FID, NPD, or FPD detector Detector temperature higher than column maximum temperature Loose column fittings Remedy: Clean injector, replace septa and liners Cut the first 10 cm of the column, if it does not help, replace the column Be sure to insert the column into the detector exactly the correct distance specified in the manual Reduce the detector temperature to the column temperature upper limit Tighten fittings accordingly

46 Baseline irregular shape: s-shaped Possible causes: Excessive column bleed during column temperature programming Oxygen contamination is decomposing the stationary phase Remedy: Reduce the upper column temperature or install a high temperature column Install oxygen filters in the carrier gas line. Check the pneumatic and inlet systems for leaks, use correct gas purity with low oxygen content

47 Baseline spiking Possible causes: Column too close to flame (when using an FID) Dirty jet or detector FID temperature too low Remedy: Lower the column to the correct position (2-3 mm below the tip of the jet) Isolate the detector from the electronics. If the spiking disappears, clean the jet and the collectors Increase the FID temperature to at least 150 ºC

48 Tailing peaks Possible causes: Poor sample transfer in the inlet Inlet temperature too low Liner contaminated Remedy: Increase the inlet temperature Check and adjust the septum purge and vent flows Alter temperature programme and check for co-elution

49 Tailing peaks Possible causes: Column degradation causing activity Column contamination Incorrect column position in inlet Blocked purge / vent line Remedy: Remove first 30 cm (1 foot) of column Re-install the column in the inlet Replace the column

50 Tailing peaks: solvent peaks Possible causes: Incorrect column position in inlet Initial oven temperature too high Septum purge flow too low and/or split vent flow too low Remedy: Reinstall the column Reduce the initial oven temperature Check and adjust the septum purge and vent flows

51 Fronting peaks Possible causes: Overloading the head of the column can lead to a fronting or wide flat-topped peak. Remedy: Injecting less Split the injection Change the column dimensions

52 Peak splitting Possible causes: Poor injection technique Poorly cut column Mixed sample solvent for splitless or on-column injections Remedy: Check injection technique Re-cut or replace column

53 Ghost peaks Causes: Contaminated carrier gas Contaminated sample/solvent Contaminated syringe/vial/injection port Inappropriate vial septa Cored septa Sample carryover Remedy: Replace the cylinder and/or the filter Check solvent compatibility with vial septa Carry out adequate clean-up of sample prior to injection Reduce injection temperature if peaks disappear use higher temperature septa Try an extended oven temperature profile

54 Trajan Scientific and Medical Trajan SGE GC Columns

55 Reproducibility SGE GC columns are manufactured from raw products Silica tubing, polyimide, stationary phase QC control at every stage Many other companies buy our tubing All columns are individually tested

56 GC column range

57 Competitor landscape Competitor Agilent Restek Phenomenex Shimadzu PerkinElmer Supelco Brands DB, DB UI, VF, HP, CP, Rxi, Rtx, MXT, Stabilwax, Zebron, Zebron Inferno SH-Rxi Elite SP, SPB, SUPELCOWAX,

58 Summary The features of a GC column The resolution equation Effects of changing GC column parameters Altering GC selectivity through temperature and phase chemistry GC column modes of interaction GC column troubleshooting

59 Trajan Scientific and Medical