CM3007 Chromatography: Theory, Sampling and Detection

1. CM3007Chromatography: Theory, Sampling and Detection Dr. Alan O’Riordan Nanotechnology Group Tyndall National Institute. Email: alan.oriordan@tyndall.ie

2. Learning Outcomes • Define and explain the theory underpinning chromatography. • To be able to explain how partition of an analyte between stationary phase and mobile phase effects separation. • To be able to identify and explain the factors influencing chromatographic separation in terms of resolution and specificity. • Identify the factors influencing different sample injection techniques and be able to discuss the advantages and disadvantages of each type. • Identify the factors influencing different analyte detection systems and be able to discuss the advantages and disadvantages of each type.

3. Classification of Chromatography Methods

4. Introduction to Chromatography Sample M obile phase Column Detector Time Separation of a mixture of components A and B by column elution chromatography. The detector signal at each stage is shown. The effectiveness of a column in separating two analytes depend in part on the relative rates at which the two components are eluted. These rates are determined by the magnitude of the equilibrium constants for the components partioned between the stationary phase and mobile phase. A mobile ⇆ A stationary

5. Mechanisms of Partition to Stationary Phase

6. Equipment Overview Sample injection Detector Mobile phase Chem station Packed column GC & HPLC Capillary column GC only

7. Partition in Chromatography • Stationary phase, mobile phase, & analyte form aternary system. • Each analyte is distributed between the two phases (in equilibrium): – Partition Coefficient – CS: concentration of analyte on the stationary phase – CM: concentration of analyte on the mobile phase

8. Using the Partition Coefficient: Plate Theory Column Column divided into theoretical pieces (plates). Analytes are partitioned between SP and MP in each plate. Separation occurs as analytes move down the column.

9. Definitions: Prototype Chromatogram

10. Factors Influencing Retention • Are those that influence distribution – Stationary phase: type & properties – Mobile phase: composition & properties – Intermolecular forces between • Analyte & mobile phase • Analyte & stationary phase – Temperature

11. Component molecule - + - + Stationary phase Intermolecular Forces I • • Based on electrostatic forces • – “Like-attracts like” or “oil and water” (similar • electrostatic properties) • • Polar/polar & non-polar/non-polar • – Molecules with dissimilar properties are not • attracted (They are NOT repelled) • • Polar retention forces • – Hydrogen bonding • (permanent dipoles) • – Dipole-Induced dipole

12. Intermolecular Forces II Polar forces (cont.): – Energy of dipole-dipole interaction : dipole moment, A: analyte, S: stationary phase – Factor of 10 variation on permanent dipole moment Factor of 104 variation on interaction energies – As r6=> mainly at the surfaces of stationary phase

13. Intermolecular Forces III (London) London’s Dispersion Forces (Van Der Waals) – Most universal interaction between molecules – Only one for non-polar species – Relatively weak – Energy of interaction: :is the polarisability, I: ionisation potential, A:analyte, S: stationary phase – As r6=> mainly at the surfaces of stationary phase How do Van Der Waal forces occur?

14. Definitions I • VR, Retention Volume • – Volume required to carry a band of component • molecules • – Primary quantity, but hard to measure! • tR, Retention Time • • FC, volume flow rate of mobile phase • – Calculated from cross section of column (dC), • average velocity of mobile phase (u), and term to • account for particle volume in columns (εT; =1 if no • particles)

15. Definitions II • u, linear velocity of the mobile phase: – L: length of the column – tM: flow time of mobile phase • Measure with non-retained component e.g. CH4 in GC • Correction for dead volume – Retention time is measured since injection – Correct for mobile phase in column at injection ( VM) t´R

16. Retardation • Recall partition coefficient • RF, retardation: • Substituting: • – 0 < RF < 1 • – If RF = 1, analyte is not retained at all • – If RF = 0, analyte is completely retained

17. Retention (or Capacity) Factor • k is like K except: – k is the ratio of total amounts, rather than of concentrations – Describes the ability of the stationary phase to retain components (same as K) – But it is a measure of actual retention properties

18. Separation Factor

19. Separation Efficiency – Peak Width Peak heights of a Gaussian peak and width as a function of standard deviation

20. Separation Efficiency – Column Efficiency

21. Initial Time, t1 Time, t2 Peak Band Broadening Processes What physical or chemical processes cause broadening of the peaks in chromatography

22. Peak Band Broadening Processes II

23. Band Broadening Van Deemter Model Recall

24. Van Deemter Model “A” Term Illustration of Eddie Diffusion

25. Van Deemter Model “B” Term Illustration of band broadening due to molecular diffusion. T3 > T2 > t1

26. Van Deemter Model “B” Term D gas≥ 104 D liquid

27. Van Deemter Model “C” Term

28. Van Deemter Model “C” Term II

29. Optimum Mobile Phase Velocity How would this plot differ for packed columns versus capillary columns?

30. Gas Chromatography:

31. Gas Chromatography – Overview • Sample is vaporised and injected onto head of a chromatography column. • Elution is effected by the flow of an inert gaseous mobile phase. • Separation is based upon the partition of the analyte between a gaseous mobile phase and a liquid phase immobilised on the surface of an inert solid (GLC) at a temperature above boiling point of analyte (multi-analyte: temperature programming). • Mobile phase does not interact with molecules of the analyte. • Eluted analyte detected by a detector and recorded by PC – Chemstation. • GC columns are either packed (with silica particles coated in stationary) or capillary in nature.

32. Sample Injection • GC column efficiency requires that the sample be of suitable size (to prevent column over loading) and be introduced as a plug of vapour. • Two common approaches include for introduction of 0.01 – 50 ml include: Microsyringe and valve loop. • The syringe technique is most common and can be used with both gas and low viscosity liquid samples by inserting the needle through a rubber septum to the column inlet port. • The region into which the needle projects must be heated in order to flash vaporise the sample. • However, overheating of the rubber septum must be avoided to prevent out gassing. • The most popular inlet for capillary GC is the split/splitless injector. • If this injector is operated in split mode, the amount of sample reaching the column is reduced (to prevent column overloading) and very narrow initial peak widths can be obtained. • For maximum sensitivity, the injector can be used in so-called splitless mode, then all of the injected sample will reach the column. • Injection may be manual or automated.

33. Split – Splitless Injection • Septum purge outlet prevents components of previous injections from entering the column and minimizes the effect of septum bleed (low flow rate ~3 ml/min). • The sample is injected into the liner region where it is completely vaporised. Mostly glass liners – zero dead volume • The sample volume is then split between the column and the split outlet. Split injection is employed to dilute the sample and prevent column overloading. Typically 1:100 split ratios are employed with 99% of sample being vented to atmosphere. • Method development: Some parameters of split/splitless injection that require optimisation, apart from instrumental design, are injector temperature, split ratio, split delay, injection volume, sample solvent and initial temperature of the column.

34. Sample Valve Injection • Sample valves are convenient for on-line gas stream analysis. • In position (a) the stream to be sampled flows through a loop of calibrated volume while the carrier gas alone passes through the column. • In position (b) the loop is placed in the carrier gas stream and the entrapped sample is swept along to the column. • Sample valves are becoming more prevalent for quantitative work employing both liquids and gases to introduce a reproducible volume of sample onto a column. • They are typically employed for smaller volumes, e.g., to prevent over loading of a column > 0.01 ml of a liquid sample is preferred volume - a precision syringe for this volume is both expensive and fragile. • Valves may also be used in split – splitless mode. (a) Sample mode (b) Inject mode

35. Pyrolysis Gas Chromatography (PGC) • A version of reaction chromatography in which a sample is thermally decomposed to simpler fragments before entering the column. 1993, 65, 827 IUPAC Compendium of Chemical Terminology • Many non-volatile solids can be decomposed thermally to produce characteristic gaseous products that can be chromatographed. • Samples are placed directly on a small coil of Pt wire where it can be heated to several hundred degrees in a few milliseconds while the carrier gas is flowing over it. • The pyrolysis products are swept directly onto the column.

36. Column Configuration Packed Columns Capillary Columns • 2 to 4 mm I.D. and 1 to 4 meters long. • Packed with a suitable adsorbent. • Mostly used for gas analysis. • Peak broadening due to zone (eddy) diffusion resulting from multitude of pathways a molecule can pass through column. • 100 mm to 500 mm I.D. and 10 m to 100 m long • Stationary phase is coated on the internal wall of the column as a film 0.2 mm to 1 mm thick • Sharper peaks – no eddy diffusion. • Up to 500,000 theoretical plates – excellent separations. • Most popular type of column in use.

37. Characteristics of Ideal GC Detector • Good stability and reproducibility. • Linear response to analytes that extends over several orders of magnitude. • Similarity in response toward all analytes. • Temperature range from room temperature to 400 ºC. • A short response time that is independent of flow rate. • Non-destructive. • High reliability and ease of use. • No one detector exhibits all of these characteristics

38. Thermal Conductivity Detector • Exploits the changes in the thermal conductivity of a gas stream brought about by the presence of analyte molecules. • The resistance of either a heated platinum wire or a heated semiconductor thermistor gives a measure of the thermal conductivity of the gas. • Twin detector pairs are typically incorporated into two arms of a Wheatstone bridge. • In the presence of a relatively small concentration of analyte a large decrease in thermal conductivity of carrier gas occurs resulting in a temperature rise in detector. • Thermal conductivities of He and H2 are ~ 6 – 10 times higher than most organic compounds. Necessitates the use of these gases as carrier gas. • Linear range of 105 and is suitable for organic and inorganic samples. • Non-destructive and allows collection of sample after detection but low sensitivity ~ 10-8 g/s analyte/gas

39. Flame Ionisation Detector • Most organic compound pyrolyse in H2-air flame and produce ions and electrons. • A potential of a few hundred volts is applied across the burner tip and a collector electrode located above the flame. • The resulting current is amplified and proportional to the number of carbon atoms in the flame. • General detector for GC. However, carbonyl, alcohol, halogen and amine groups yield few electrons. Also insensitive to H20 CO2 SO2 NOX. • Large linear response range (~ 107) and low noise (once detector has settled). Needs to be burning 24 hours before analysis. • Exhibits very high sensitivity ~ 10-13 g/s of analyte/second Collector electrode Insulator Connector nut Air H2-air flame Grounded jet H2 Inside oven wall Exit end of column

40. Advantages and Disadvantages of GC • Fast analysis • Typically minutes (even sec.) • High Resolution • Record N~1.3 x 106 • Sensitive detectors (easy ppm, often ppb) • Highly accurate quantification (1-5 % RSD) • Automated systems • Non-destructive • Allows online coupling to Mass Spec. • Small sample (mL) • Reliable and relatively simple • Low cost (~€20,000) • Limited to volatile samples • T limited to ~ 380 °C • Need Pvap ~ 60 Torr at that temperature • Not suitable for thermally labile samples • Some samples may require extensive preparation • Requires spectroscopy (usually MS) to confirm peak identify

41. CM3007Gas Chromatography: Method Development and Validation Dr. Alan O’Riordan Nanotechnology Group Tyndall National Institute. Email: alan.oriordan@tyndall.ie

42. Learning Outcomes • Be able to differentiate between different GC column types. • Explain how mobile phase flow rate, temperature and type of column can affect column resolution and sensitivity. • Determine/identify suitable stationary phase for analytes. • Distinguish between different quantitative approaches to GC. • Specify and explain the eight requirements necessary for method validation. • Identify and explain the different approaches to analyte sampling and injection. • Identify two application areas for GC.

43. Choice of GC Columns

44. Packed GC Columns

45. Open (capillary) GC Columns

46. Column Type Vs Separation

47. Comparison of Columns

48. Effect of Column Diameter & Film Thickness

49. Optimum Mobile Phase Velocity

50. Effect of Column, Film and Carrier Gas