Chemistry 311: Instrumental Analysis Topic 4: Basic Chromatography. Chemistry 311: Instrumental Analysis Topic 4: Basic Chromatography

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Percival Hudson
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Key Chromatographic techniques: Column Chromatography Liquid Chromatography HPLC Ion Chromatography Gas Chromatography Packed Column Capillary Column Supercritical

1 Introductory Theory, Basic Components, Qualitative and Quantitative applications. HPLC, GC, Ion Chromatography. Rouessac Ch. 1-7 Winter 2011 Page 1 Chromatography: The separation of analytes based on differences in partition between a mobile phase and an immiscible stationary phase. Key Chromatographic techniques: Column Chromatography Liquid Chromatography HPLC Ion Chromatography Gas Chromatography Packed Column Capillary Column Supercritical Fluid Chromatography Planar Chromatography Thin Layer Chromatography (TLC) Related Non-chromatographic techniques: Electrophoresis Capillary Electrophoresis Ion Mobility Detectors Mikhail Tsvet ( ): Page 2 1

Solutes that bind to the stationary phase more strongly are retained longer Page 3 The manner in which the analyte can be retained on (or in) the stationary

2 In general the sample mixture is applied to the column (or stationary phase) and then washed through the column by the mobile eluent. Rate at which the solute is washed from the column is dependant on the average time it spends partitioned into the mobile phase or visa versa. Solutes that bind to the stationary phase more strongly are retained longer Page 3 The manner in which the analyte can be retained on (or in) the stationary phase is quite variable. Examples include; Note: From this it is clear why electrophoresis is not a form of chromatography as partitioning into a stationary phase is not the basis of separation Page 4 2

3 Page 5 The concentration of the analyte can be measured as a function of time or volume interchangeably to obtain a valid chromatogram The degree of separation can be improved either by increasing the degree of band separation or by decreasing the band spread Page 6 3

4 Distribution Constants: K is the distribution constant or partition ratio Retention time (t r ) is the time it takes a analyte to reach the detector after it is injected. Time for an unretained peak takes to reach the detector is the dead time (t m ) Linear velocity of the solute (v) and the mobile phase (u) is; Page 7 Retention factor or Capacity factor (k A ): Describes the migration rates of solutes on a column. Selectivity factor (α): An indication of the relative selectivity of a column for different analytes. A and B selected such that α is always greater than unity. Plate height (H): An indication of the efficiency of column in terms of length of column required for a degree of separation. H is inter-related to the length (L) of the column. Therefore, the number of theoretical plates per column (N) is also important. In order to measure H, the gaussian nature of a peak is used, from this H can be related to the variance (σ 2 ). Page 8 4

5 Since chromatograms are measured by time. Need to convert from the time scale to cm. The standard deviation of a time based chromatographic is expressed by τ rather than σ to avoid confusion. Page 9 Since H is related to mass-transfer rates, plate height (H) must be a function of the rate of flow. Page 10 5

Page 11 The Multipath term (A): In a packed column the solute can travel by many routes, some of which are longer than others. Also called eddy diffusion.

6 van Deemter equation: H = A + B/u + Cu = A + B/u + (C S + C M )u Where; u = linear velocity (cm/s) A = multiple flow paths B = longitudinal diffusion C = mass transfer between phases, C S in stationary phase, C M in mobile phase. Page 11 The Multipath term (A): In a packed column the solute can travel by many routes, some of which are longer than others. Also called eddy diffusion. A = 2λ d p Where; λ is a proportionality constant d p is diameter of packing particles The Longitudinal Diffusion term (B/u): Diffusion that occurs due to the concentration gradient in the mobile phase. Where; γ = obstruction factor (~ 0.6 packed, unpacked unity) D m = mobile phase diffusion coefficient Page 12 6

Mass-transfer Coefficients (C S and C M ): The chromatographic process is sufficiently fast that equilibrium between the analyte in the stationary phase and mobile phase does not occur.

7 Mass-transfer Coefficients (C S and C M ): The chromatographic process is sufficiently fast that equilibrium between the analyte in the stationary phase and mobile phase does not occur. Furthermore, the thickness of each of these layers means there is a statistical variation in transition time in each layer. Page 13 Summary of Effects of van Deemter equation terms: Page 14 7

8 Column Resolution (R S ): A quantative measure of the ability to separate two analytes; Page 15 Page 16 8

9 Page 17 Page 18 9

10 Page 19 Gas Chromatography: Normally refers to gas-liquid rather than gas-solid. All the general chromatographic concepts discussed previously apply, although, temperature and pressure effects are more significant. Normally, measure average gas flow with bubble meter and then converted to volume Page 20 10

11 Carrier Gas: Inert usually, He, N 2, or H 2 Flow controlled by two stage regulators and flow controllers. Inlet P PSI > ambient Flow mls/min packed Flow 1 25 mls/min cap. Page 21 Injection Systems: Page 22 11

12 11/3/11 Page 23 Columns: The column is the device used to separate analytes. Therefore, column properties and characteristics are key to any chromatographic technique Page 24 12

13 Page 25 Page 26 13

14 Page 27 Detectors: Page 28 14

Detectors: Flame Ionization Detector (FID): Monitors changes in

Signal is proportion to the number of Carbon atoms entering the

Signal is directly proportion to mass over several orders of

Electron Capture Detector (ECD): Ionization of carrier gas or

15 Detectors: Flame Ionization Detector (FID): Monitors changes in ion current due to flame induced pyrolysis. Signal is proportion to the number of Carbon atoms entering the flame. Signal is directly proportion to mass over several orders of magnitude. Electron Capture Detector (ECD): Ionization of carrier gas or make-up gas occurs due to high energy β particles (e-) from a Ni-63 foil. Current is monitored. The current decreases rapidly with high electron affinity compounds pass through Page 29 Page 30 15

Volatile Components: Headspace analysis: Sample is

GC. Purge and Trap methods: See Following page

16 Volatile Components: Headspace analysis: Sample is sealed in a vial, heated and the headspace sampled using a gas tight syringe. Alternately headspace vapor transferred directly to GC. Purge and Trap methods: See Following page Solid Phase Micro-Extraction: See Following pages Page 31 Headspace: Page 32 16

Solid Phase MicroExtraction (SPME): Page 33 Solvent Extraction Methods Method 3500B: Organic Extraction and Sample Preparation Method 3510C: Separatory Funnel Liquid-Liquid Extraction Method 3520C:

17 Solid Phase MicroExtraction (SPME): Page 33 Solvent Extraction Methods Method 3500B: Organic Extraction and Sample Preparation Method 3510C: Separatory Funnel Liquid-Liquid Extraction Method 3520C: Continuous Liquid-Liquid Extraction Method 3535: Solid-Phase Extraction (SPE) Method 3540C: Soxhlet Extraction Method 3541: Automated Soxhlet Extraction Method 3545: Pressurized Fluid Extraction (PFE) Method 3550B: Ultrasonic Extraction Method 3561: Supercritical Fluid Extraction of PAH Method 3580A: Waste Dilution Clean-up methods Method 3600C: Cleanup Method 3610B: Alumina Cleanup Method 3620B: Florisil Cleanup Method 3630C: Silica Gel Cleanup Method 3640A: Gel-Permeation Cleanup Method 3650B: Acid-Base Partition Cleanup Method 3660B: Sulfur Cleanup Method 3665A: Sulfuric Acid/Permanganate Cleanup Page 34 17

B) Continuous Liquid-Liquid Extraction System: Usually designed to provide

18 A) Typical Soxhlet Extractor: Usually designed to provide continuous extraction of a solid material such as soil. B) Continuous Liquid-Liquid Extraction System: Usually designed to provide continuous extraction of a water sample, design is dependent on density of extracting liquid. Page 35 Extraction Distribution Law: Page 36 18

Solid Phase Extraction: Simple low efficiency chromatography step to isolate analyte compounds from matrix and some interferences. Clean-up Methods: Numerous Chromatographic based clean-up techniques.

19 Solid Phase Extraction: Simple low efficiency chromatography step to isolate analyte compounds from matrix and some interferences. Clean-up Methods: Numerous Chromatographic based clean-up techniques. Mostly designed to remove macromolecules, lipids etc. Acid or Base digestion may also be used for very stable species such as PCB s and Dioxin, etc. Sulfur Clean-up: Elemental sulfur is encountered in many sediment samples The solubility of sulfur in various solvents is very similar to the organochlorine and organophosphorus pesticides. Page 37 Two techniques for the elimination of sulfur are; 1) the use of copper powder Concentrate the sample to exactly 1.0 ml or other known volume. Transfer 1.0 ml of the extract to a calibrated centrifuge tube. Add approximately 2 g of cleaned copper powder to the centrifuge tube. Vigorously mix the extract and the copper powder (at least 1 min) Allow the phases to separate. Draw off the extract. 2) the use of tetrabutylammonium sulfite (TBA). Concentrate the sample extract to exactly 1.0 ml or other known volume. Transfer 1.0 ml a 50 ml clear glass bottle or vial Rinse the concentrator tube with 1 ml of hexane, add rinsings. Add 1.0 ml TBA sulfite reagent and 2 ml 2-propanol, shake for 1 min. If colorless, or if initial color is unchanged, and clear crystals observed then sufficient sodium sulfite is present. If precipitated sodium sulfite disappears, add more crystalline sodium sulfite Add 5 ml organic free reagent water and shake for at least 1 min. Allow sample to stand for 5-10 min. Transfer the hexane layer (top) to a concentrator tube and concentrate the extract to approximately 1.0 ml Page 38 19

high boiling material that may result in; errors in quantitation; false positives or negatives,

20 Page 39 Analytical Steps (cont.): Clean-up: The purpose of applying a cleanup method to an extract is to remove interferences and high boiling material that may result in; errors in quantitation; false positives or negatives, deterioration of expensive capillary columns; and, instrument downtime caused by cleaning and rebuilding of detectors and ion sources. (See EPA Method 3600C: Cleanup) Page 40 20

21 HPLC Systems: Originally called High Pressure Liquid Chromatography, now referred to as High Performance Liquid Chromatography. Useful for a wide range of compounds Page 41 HPLC Instrumentation: Page 42 21

22 Pumping Systems: The requirements for an HPLC pumping system are; Generation of pressures of to 6000 psi (lbs/in 2 ), Pulse-free output, Flow rates from 0.1 to 10 ml/min, Flow control and flow reproducibility of 0.5% relative or better, Corrosion-resistant components (seals of stainless steel or Teflon). Three types of pumps: Reciprocating pumps: Most common, 90% of applications. Tend to produce pulsed output, need to be dampened. High Pressure 10,000 psi Syringe or displacement-type pumps: Large syringe like pump with a mechanically driven plunger. The output is relatively pulse free but limited in volume. Pneumatic or constant pressure pumps: Sample container a collapsible container and pressurized by an inert gas. Limited capacity, pressure. Page 43 Reciprocating pumps: Most common, 90% of applications. Tend to produce pulsed output, need to be dampened. High Pressure 10,000 psi Page 44 22

Sample Injection Systems: The limiting factor in the precision of liquid chromatographic is the reproducibility with which samples can be introduced onto the column packing.

23 Sample Injection Systems: The limiting factor in the precision of liquid chromatographic is the reproducibility with which samples can be introduced onto the column packing. The most widely used method of sample introduction in liquid chromatography is sampling loops, see below. Often have interchangeable loops providing a choice of sample sizes from 5 to 500 µl. Sampling loops of this type permit the introduction of samples at pressures up to 7000 psi with precisions of a few tenths percent relative. Page 45 Solvent Degassing: One critical parameter is the quality of the solvents used. Solvents must be free of particulate, contaminants and dissolved gases. Dissolved gases come out of solution and form micro-bubbles which can interfere with accurate flow and become lodged in detector cells, creating baseline instability. A number of ways to reduce or eliminate the problem are available. External Vacuum Degassing: A simple and effective form of degassing is to hold a flask of mobile phase under a vacuum while agitating the contents using a stirrer or an ultrasonic bath. Helium Sparging: Helium sparging is a simple technique: a helium tank is connected to the solvent reservoirs of the chromatograph, and helium is bubbled into the eluent. Because helium has very low solubility in most solvents, it displaces the more common gases from the mobile phase. This approach has two limitations; 1) large quantity of expensive helium gas required, and the potential exists for changing the composition of the premixed solvents by selectively volatilizing the more volatile solvent. On-line Degassing: A vacuum is drawn on semipermeable tubes through which the eluents run. The vacuum draws air from the flowing solvents and discards it to waste. Page 46 23

24 Columns: The column is the heart of the chromatograph, providing the means for separating a mixture into components. The selectivity, capacity, and efficiency of the column are all affected by the nature of the packing material or the materials of construction. Stationary Phases: Most modem HPLC packings are microparticles of varying size, shape, and porosity. Silica gel is the main stationary phase for adsorption chromatography, although the metal oxides, and polymeric adsorbents, such as polystyrene divinylbenzene have also been used. Silica packings can withstand the high pressures, is abundant, inexpensive, and available in a variety of shapes, sizes, and degrees of porosity. In addition, functional groups can be readily bonded to the silanols, and the chemistry of the bonding reactions are well understood. Instable at low,high ph. Page 47 Requirements for an Ideal HPLC Column: 1. Particles should be spherical, in particle diameters ranging from 3 to 10 µm. 2. Withstand pressures of psi and not swell or shrink with eluent. 3. Particles should have porosity in the range 50 to 70% (80% for size-exclusion) 4. No pores smaller than 60 Å in diameter with a uniform pore size distribution. 5. Range of mean pore diameters of Å 6. The internal surface of the material should be homogeneous. 7. Able to modify surface to provide a range of surface functionalities. 8. Packing should be inert under all conditions of ph and eluent composition. 9. The physicochemical characteristics should be reproducible 10. Material should be readily available and relatively inexpensive, and its chemical behavior should be well understood. Page 48 24

25 HPLC Classifications: Isocratic: Eluent has a constant composition through out the analysis. This simplifies system requirements and set-up, however, good resolution occurs only for a limited set of analytes. Gradient systems: Eluent composition changes during the collection of a chromatograph. A series of solvent systems can be mixed together and this mix varied in a gradual manner, effectively altering the solvation ability of the mobile phase. Generally, two-binary, three-ternary or four-quaternary, solvents systems can be mixed. However, binary systems are the most common. Page 49 Reverse-Phase and Normal-Phase Packings: In early LC work, columns contained water or triethyleneglycol supported on silica or alumina particles were commonly used. Common mobile phases were non-polar solvents such as hexane. For historic purposes this type of system was called Normal-Phase. Reverse-Phase chromatography refers to systems where the stationary phase is non-polar and the mobile phase is non-polar (water, methanol, or acetonitrile). Page 50 25

Detectors: This is weakest aspect of HPLC instrumentation. No adequate universal detector. A high percentage (>70%) of analysis uses UV based detection.

26 Detectors: This is weakest aspect of HPLC instrumentation. No adequate universal detector. A high percentage (>70%) of analysis uses UV based detection. Page 51 Fixed Wavelength UV Detectors: The most common source is the low pressure Mercury lamp. This device emits 90% of it s light at 254 nm. Filters can be used to isolate this discrete line. Many organic analytes absorb strongly at this λ. Variable Wavelength and Diode-Array UV Detectors: These permit convenient variation in the measured λ or monitor a range of λ simultaneously. Much more convenient, yet quite expensive (~$20,000 for diode array). Page 52 26

27 Partition, Ion-Pair and Ion-Exchange Chromatography: Most of the concepts discussed so far have been based on Partition Chromatography, or the partitioning of analytes between the mobile and stationary phases due to relative solubilities. In Ion-Pair Chromatography, the analyte is an ion that forms a neutral ion-pair complex with a counter ion in the mobile phase. This neutral ion-pair complex can then partition into the stationary phase. In Ion-Exchange Chromatography the analyte ion is exchanged for a ion of similar charge which is paired to the solid support. In this separation is not based on partition solubility but rather ion exchange equilibria. Page 53 For Cation Exchange: Page 54 27

Ion Exchange Packings: Page 55 Conductivity Detection for Ion Chromatography: In order for this relatively inexpensive but sensitive detection technique to be applicable, the signal due to the eluent

28 Ion Exchange Packings: Page 55 Conductivity Detection for Ion Chromatography: In order for this relatively inexpensive but sensitive detection technique to be applicable, the signal due to the eluent must be accounted for or eliminated. Eluent Suppressor Columns: A column packed with a resin that converts the eluent ions to a molecular species with low conductivity ie., for HCl New Suppressor Columns can are regenerated continuously. Page 56 28

Page 57 THIN-LAYER CHROMATOGRAPHY (TLC): Planar chromatographic methods include thin-layer chromatography (TLC) and paper chromatography (PC), and electrochromatography.

29 Page 57 THIN-LAYER CHROMATOGRAPHY (TLC): Planar chromatographic methods include thin-layer chromatography (TLC) and paper chromatography (PC), and electrochromatography. Each makes use of a flat, relatively thin layer of material that is either self supporting or is coated on a glass, plastic, or metal surface. The mobile phase moves through the stationary phase by capillary action, sometimes assisted by gravity or an electrical potential. Planar chromatography is sometimes called twodimensional chromatography. An important use of thin-iayer chromatography, is to serve as a guide to the development of optimal conditions for performing separations by column liquid chromatography. The advantages of following this procedure are the speed and low cost of the exploratory thin-iayer experiments. Page 58 29

30 Sample applied as a small spot near an edge of plate (ie. capillary tube). Evaporate the solvent and then develop by placing edge of plate into solvent. Solvent is drawn into TL-Plate and carries solute with it. To see spots spray with solution of iodine, sulfuric acid or related. Page 59 Page 60 30

31 Carrier Gas: Inert usually, He, N 2, or H 2 Flow controlled by two stage regulators and flow controllers. Inlet P PSI > ambient Flow mls/min packed Flow 1 25 mls/min cap. Page 61 Carrier Gas: Inert usually, He, N 2, or H 2 Flow controlled by two stage regulators and flow controllers. Inlet P PSI > ambient Flow mls/min packed Flow 1 25 mls/min cap. Page 62 31

Introduction to Chromatographic Separations

Introduction to Chromatographic Separations Analysis of complex samples usually involves previous separation prior to compound determination. Two main separation methods instrumentation are available: