Topic 8. Chromatography

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1 Topic 8 Chromatography

2 Separations

3 Analytical Separation 1. A substance that affects an analytical signal or the background is called an interference or an interferent. 2. Separations isolate the analyte from potentially interfering constituents. 3. The internal standard and standard addition methods can sometimes be used to compensate for or to reduce interference effects. 4. Separations can be complete or partial and always requires energy because the reverse process, mixing at constant volume, is spontaneous and accompanied by an increase in entropy.

5 Separation methods To eliminate or reduced interferences for complex mixtures before quantitative analysis.

6 Separation by extraction Solvent Extraction : Aaq Aorg transfer of a solute from phase 1 phase 2 S (in phase1) S (in phase 2) Distribution constant K s 2 s 1

7 Solid-phase extractions or liquid-solid extraction Determining organic constituents in drinking water by methods approved by the Environmental Protection Agency. Advantages: reducing extraction time and lowering solvent use.

Separating ions by ion exchange In the ion-exchange process, ions held on an ion-exchange resin are exchanged for ions in a solution brought into contact with the resin.

8 Separating ions by ion exchange In the ion-exchange process, ions held on an ion-exchange resin are exchanged for ions in a solution brought into contact with the resin. Strong-acid-type: sulfonic acid groups (--SO3-H+) attached to the polymeric matrix. Strong-base anion: quaternary amine [--N(CH3)3+OH-] groups, while weak-base types contain secondary or tertiary amines.

9 1) Structures Ion-Exchange method Cation exchangers: -SO3 -, -CO2 - Anion exchanger: -NR3 +

10 Ion-Exchange method 2) Ion-exchange selectivity a) R - K + + Li + R - Li + + K + selectivity coefficient: K R Li K R K Li

11 Ion-Exchange method b) Hydrated radius : hydrated radius or Ionic charge bind to ion-exchange resin order of selectivity:

12 Applications of Ion-Exchange method 3) What is deionized water? 4) Preconcentration trace analysis [s] is extremely low important for environmental problem

13 Ion-Exchange Chromatography Metals in natural waters can be preconcentrated with a cation-exchange column. The cations can then be displaced into a small volume of solution by eluting the column with concentrated acid

14 Principles of Chromatography Chromatography is the most powerful tool for separating & measuring the components of a complex mixture. Quantitative & qualitative analysis

15 What is Chromatography? Chromatography : same as extraction a) One phase: held in place stationary phase. solid material (packing material) b) Another phase : fluid phase mobile phase. sample gas (GC) liquid (LC)

16 What is Chromatography? c) Invented by M.Tswett in 1903 (adsorption chromatography ) d) Elution: a process in which solutes are washed through a stationary phase by the movement of a mobile phase & is always (100%) dilution. e) Eluent is a solvent used to carry the components of a mixture through a stationary phase.

17 Elution Chromatogram

18 Migration rates of solutes A solute equilibrates between a mobile and a stationary phase. The more it interacts with the stationary phase, the slower it is moved along a column. X(mobile) X(stationary) Kc = [X]s / [X]m Solutes with a large Kc value will be retained more strongly by the stationary phase.

19 Column chromatography the stationary phase is held in a narrow tube, and the mobile phase is forced through the tube under pressure or by gravity.

20 Types of Chromatography Divided into categories on the basis of the mechanism of interaction of the solute v.s. the stationary phase.

21 Types of Chromatography polar s.p. for GC & LC for GC

22 Types of Chromatography resin-so 3 - gel filtration resin-n(ch 3 ) 3 + by size

23 Types of Chromatography the most selective one ph, and ionic strength

24 How do we describe a chromatogram 1) Chromatogram : A graph showing the detectors response as a function of elution time : band s shapes, position, resolution.

25 How do we describe a chromatogram 2) For individual band : a) Retention time (t r ) : the time needed after injection for an individual solute to reach detector. b) An ideal chromatographic peak Gaussian shape. w ½ = 2.35σ, w = 4σ

26 How do we describe a chromatogram 3) For pairs of bands a) Efficiency : two factors contribute to how well components are separated : the widths of the peaks : the wider the peak, the poorer separation. the spacing in time : the further apart, the better separation.

27 How do we describe a chromatogram

28 How do we describe a chromatogram b) Theoretical plates (N): (from distillation) the more plates on a column, the more equilibration steps, and the better the separation. Number of plates on column : N = 16(t R /w) 2 Plate height : H = L/N The smaller plate height narrower peaks better separation

29 c) Resolution (Rs) B A A R B R B A B A s W W t t W W Z W W Z R ] ) ( ) 2[( L 2Rs the s.p. length of 2 2 Rs For quantitative analysis, How do we describe a chromatogram

30 How do we describe a chromatogram d) Improved separations (1)increase in band separation. (2)decrease in band widths.

31 Theory of bands broadening 1) Why broadening? a) diffusion b) slow equilibration of solute between the m.p and s.p. a)irregular flow paths.

32 Theory of bands broadening 2) Longitudinal diffusion : the faster the flow the less a band spends in column. the less time for diffusion. broadening 1 u

33 Theory of bands broadening 3) solute requires time to equilibrate between phases. m.p. s.p. (s.p. m.p.) with temp. broadening u Can t equilibrate rapidly enough.

34 Theory of bands broadening 4) A Separation Has an Optimum Flow Rate The rate of mass transfer between phases increases with temperature.

35 Theory of bands broadening 5) Multiple paths

36 Theory of bands broadening 6) Plate height equation

37 Theory of bands broadening Plate height equation

38 Theory of bands broadening Effect of particle diameter GC LC

39 Theory of bands broadening 7) open tubular columns Packed column (A, B, C 0 in van Deemter s eqn.) Open tubular column (A = 0 in van Deemter s eqn.) resolution ( H & column length ) sample capacity ( less s.p.)

40 Theory of bands broadening 8) Funny shapes polar solute OH OH (CH3)3SiO OSi(CH3)3 Si O Si Si O Si s.p. silanization

41 Homework 31-5 (a) ~ (e)

42 GC & LC

43 Gas Chromatography 1. Schematic diagram capillary columns

44 Gas Chromatography 2. Columns : open tubular columns

46 Gas Chromatography A) Two types: m.p.(gas) - s.p. 1) s.p.: solid(using adsorption)ex: SiO 2 column ages: Si-O-H cause tailing peak. 2) s.p.: liquid(glc, using partition) a range of polarities, like dissolves like Decrease thickness of stationary phase leads to a) Resolution (H ) b) t r c) Sample capacity

47 Gas Chromatography S.P. General structure:

49 Gas Chromatography B) The effects of column polarity on separation Like dissolves like (a) S.P: nonpolar, b.p. dependent (b) S.P: polar

50 Gas Chromatography packed column vs. open tubular column higher resolution lower sample capacity

51 Gas Chromatography 3. Temperature programming temp of column v.p. solute, t r sharpens peaks isothermal : constant temp. temp. programming (gradient) : raise the column temp. during the separation.

52 Column temperature is an important variable that must be controlled for precise work. The optimum column temperature depends on the boiling point of the sample and the degree of separation required.

53 Gas Chromatography (a) Isothermal and (b) programmed temperature chromatography of linear alkanes through a packed column with a nonpolar stationary phase.

54 4. Carrier Gas Gas Chromatography

55 Gas Chromatography 5. Sample Injection 1) gasses, liquids, or solids vaporized, not decomposition 2) injection time bands broader 3) injected by syringe (manual or automatic injection)

56 Gas Chromatography Injection port operation for (a) split, (b) splitless, and (c) oncolumn injection into an open tubular column.

57 Gas Chromatography 5. Detectors Qualitative analysis : mass spectrometer, IR Quantitative analysis : area of a chromatographic peak.

58 Table 32-1 p892

59 Mass Spectrometry Detector - A mass spectrometer is the single most versatile detector. - Total Ion Chromatogram (TIC): The ion abundance in each spectrum can be summed and plotted as a function of time to give a total-ion chromatogram. - Selected ion monitoring (SIM): A single m/z value can be selected and monitored throughout the chromatographic experiment - Selected reaction monitoring (SRM) = tandem mass = MS/MS - Multiple reaction monitoring (MRM)

60 Ex: GC-MS, TIC, SIM Typical outputs for GC/MS: GC/TIC Ex: GC/SIM MS for m/z 168, R t 10.46

61 Information in a mass spectrum Rxn : CH 3 (CH 2 ) 2 CH 2 OH + Br - CH 3 (CH 2 ) 2 CH 2 Br 1 Butanol 1 Bromobutane

62 Information in a mass spectrum Fragmentation Patterns CH 3 15 CH 2 14 Br 79 C 4 H 9 79 Br % C 4 H 9 81 Br +

63 Isotope dilution: Caffeine as example

64 Caffeine (CH 2 D) as an internal standard Why does the chromatogram show a single peak? Why was m/z used for IS? Why is an isotopic variant a good IS?

65 Ask yourself 1. What nominal mass is being observed in SIM? Why are only three peaks observed? 2. Suggest a structure for m/z 73 for MTBE and TAME., m/z 87 for ETBE and TAME MTBE ETBE TAME

66 Applications of gas-liquid chromatography Qualitative Analysis: GC is widely used to establish the purity of organic compounds. Contamination if present: additional peaks in chromatogram. Evaluating the effectiveness of purification procedures. Tr is useful for identifying components in mixtures. Spike experiment, no new peaks appear, and enhancement observed. Duplicated on different columns and at different temperatures. Although a chromatogram may not lead to positive identification of the species in a sample, it often provides sure evidence of the absence of species.

67 Applications of gas-liquid chromatography Quantitative Analysis: Figure illustrates the point that computers and humans may not choose the same baseline for measuring area.

68 Applications of gas-liquid chromatography Quantitative Analysis 1. Quantitative GC is based on comparison of either the height or the area of an analyte peak with that of one or more standards. 2. If conditions are properly controlled, both of these parameters vary linearly with concentration. 3. Peak area is independent of the broadening effects. 4. Calibration with Standards

69 Applications of gas-liquid chromatography The Internal Standard (IS) Method 1. The highest precision for quantitative GC is obtained using ISs because the uncertainties introduced by sample injection, flow rate, and variations in column conditions are minimized. 2. A carefully measured quantity of an IS is introduced into each standard and sample and the ratio of analyte peak area (or height) to IS peak area (or height) is used as the analytical parameter.

70 Applications of gas-liquid chromatography IS Method IS is known amount of a compound, different from analyte, that is added to an unknown. To use an IS, we prepare a known mixture of standard and analyte and measure the relative response of the detector to the two species. In Figure, the area under each peak is proportional to the concentration of each compound injected into the column. [X] and [S] are the concentrations of analyte and standard after they have been mixed together.

71 Example : Using an Internal Standard In a chromatography experiment, a solution containing M X and M S gave peak areas of A x =423 and A S =347. To analyze the unknown, 10.0 ml of M S were added to 10.0 ml of unknown, and the mixture was diluted to 25.0 ml in a volumetric flask. This mixture gave the chromatogram in Figure, with peak areas A x =533 and A S =582. Find the concentration of X in the unknown.

72 SOLUTION: Because X was diluted from 10.0 to 25.0 ml when the mixture with S was prepared, the original concentration of X in the unknown was (25.0/10.0)( M)=0.143 M.

73 Homework 32-5, 32-6, 32-7, 32-18, Internal standard. A known mixture of compounds C and D give the following chromatography results: Compound C g/ml) Peak area(cm 2 ) C D A solution was prepared by mixing 1.23 mg of D in 5.00 ml with ml of unknown concentration of C ( g/ml) in the unknown.

74 High-Performance Liquid Chromatography 1. open, gravity-feed column for LC. 2. closed column (under high pressure) packed with micron-size particles. (HPLC) 3. Type of stationary phase : partition, or liquid-liquid, chromatography; adsorption, or liquid-solid, chromatography; ion-exchange, or ion chromatography; size-exclusion chromatography; affinity chromatography; and chiral chromatography.

75 Effect of particles size of packing & flow rate 1. Through a closed column, and needs high pressure. 2. s.p. particles size microporous particles of silica with diameters of um s.p. m.p. faster, i.e. C in van Deemter eqn. resolution

76 Methods chosen based on: 1. solubility 2. molecular mass. Application of LC

77 the high pressures make the equipment for HPLC tends to be more elaborate and expensive. Instrumentation

78 Instrumentation

79 300-L preparative column: for purification

80 Eluent strength compete with for binding on s.p. the more strongly bind to s.p. eluent strength

81 Eluent strength Elution The more polar solvent eluent strength t r Gradient elution : increased the eluent strength during the separation in liquid chromatography.

82 Solvents a) Isocratic elution : elution with single solvent or a constant solvent mixture b) Gradient elution : solvent is changed continuously from a weak eluent strength to a strong eluent strength by mixing more and more of a strong solvent to a weak solvent during the chromatography.

83 Solvents

84 Column 1. Analytical Columns: 5 to 25 cm in length and have inside diameters of 3 to 5 mm. 2. Micro columns: packed with 3- or 5-mm particles, have the advantage of speed and minimal solvent consumption. 3. Precolumns: (a)scavenger column: between the mobile phase reservoir and the injector is used for mobile-phase conditioning (b)guard column: between the injector and the analytical column and prevents impurities, such as highly retained compounds and particulate matter, from reaching and contaminating the analytical column

85 HPLC Detectors The most widely used detectors for LC are based on absorption of ultraviolet or visible radiation.

86 Partition chromatography In this type of chromatography, the stationary phase is a second liquid that is immiscible with the liquid mobile phase. Bonded-Phase Packings: The R group is often a straight chain octylor octyldecyl-group. Other organic functional groups that have been bonded to silica surfaces include aliphatic amines, ethers, and nitriles as well as aromatic hydrocarbons.

87 Stationary phase Normal-phase chromatography : polar s.p. and less polar solvent. Eluent strength is increased by adding a more polar solvent. b) Reversed-phase chromatography : low-polarity s.p. and polar solvent. Eluent strength is increased by adding a less polar solvent.

88 Choice of Mobile and Stationary Phases

91 Ex: Figure Isocratic HPLC separation of a mixture of aromatic compounds at 1.0 ml/min on a cm Hypersil ODS column (C 18 on 5-μm silica) at ambient temperature (~22 ): (1) benzyl alcohol; (2) phenol; (3) 3, 4 -dimethoxyacetopheneone; (4) benzoin; (5) ethyl benzoate; (6) toluene; (7) 2,6-dimethoxytoluene; (8) o-methoxybiphenyl. A : KH 2 PO 4 (aq) B: CH 3 CN (l)

92 The gradient can be used to resolve all peaks by reducing the time from 2 h to 38 min.

93 Ask yourself Nonpolar aromatic compounds were separated by HPLC on a bonded phase containing [-(CH2)17CH3] covalently attracted to silica. Eluent was 65% vol% methanol in water. How would Tr be affected if 90% methanol were used instead? When you try separating an unknown mixture by reversed-phase chromatography when 50% acetonitrile- 50% water, the peaks are eluted between 1 and 3 min and they are too close together. Should you higher or lower % of acetonitrile in next run?

94 Ion-Exchange method-review Structures for cation and anion exchange Cation exchangers: -SO3 -, -CO2 - Anion exchanger: -NR3 +

95 Ask yourself 1. Haxanoic acid and 1-aminohexane, adjusted to ph 12 with NaOH, were passed through a cation-exchange column load with NaOH at ph12. State the principle species that will be eluted and the order in which they are expected. What if adjusted to ph 3 with HCl? 2. Consider a negatively charged protein adsorbed on anion-exchange gel at ph 8. (a) How will a gradient from ph 8 to some lower ph? (b) How would a gradient of increasing [NaCl] at constant ph?

96 Ion Chromatography (1) a high-performance version of ion-exchange chromatography, with a key modification that removes eluent ions before detecting analyte ions. (2) in semiconductor industry: to monitor anions & cations level at 0.1-ppb levels in deionized water. (3) in environmental analysis:

97 Ion Chromatography (4) Anions are separated by ion exchange & detected by their electrical conductivity. Problem : It is difficult to detect the conductivity change when analyte ions are eluted. Suppressed-ion anion chromatography : Remove the unwanted electrolyte prior to conductivity measurement.

98 Ion Chromatography HCl [con]? time cation separator column (H+) [con]? time suppressor column OHreplaces anion

Molecular Exclusion Chrom. Molecules are separated according to their SIZE. 1. Gel filtration: a type of sizeexclusion chromatography in which the packing is hydrophilic.

99 Molecular Exclusion Chrom. Molecules are separated according to their SIZE. 1. Gel filtration: a type of sizeexclusion chromatography in which the packing is hydrophilic. It is used to separate polar species. 2. Gel permeation: a type of sizeexclusion chromatography in which the packing is hydrophobic. It is used to separate nonpolar species.

100 Affinity Chromatography Isolate a single compound from a complex mixture. a. specific binding to s.p. b. unbind : change ph or change ionic strength Interaction between: enzymes / substrates antibodies / antigens receptors / hormones

101 Chiral chromatography Optical isomers: nonsuperimposable mirror images of each other. It is important for drug industry. D- & L-amino acids Ex: for ant-inflammatory drug Naproxen

102 Chiral chromatography

103 Comparison of HPLC & GC

104 Homework 33-3, 33-4, 33-5, 33-6, 33-7, 33-9

105 Capillary Electrophoresis

106 What is Capillary Electrophoresis? Electrophoresis is a separation method based on the differential rates of migration of charged species in an applied dc electric field.

107 Electropherogram: Sensitivity capillary electrophoresis : extremely high resolution in a narrow capillary tube (only B term in the van Deemter eqn.) No s.p. C = 0 Open tubular column A = 0

108 What is Capillary Electrophoresis? (1) The greater the charge on the ion, the faster it migrates. (2) The greater the size of the molecule, the slower it migrates. (3) Different ions migrate at different speeds, so they separate.

109 What is Capillary Electrophoresis? (1) Two processes operate in capillary electrophoresis: (a) electrophoresis: the migration of ions in an electric field cation cathode anion anode (b) electroosmosis: pump the entire solution through the capillary anode cathode

110 What is Capillary Electrophoresis? (2) Why electroosmosis? (a)wall is covered with silanol, ph > 2, Si-OH Si-O - Electric double layer (Diffuse part of the double layer ~1 nm) (b) Electric field flow (c) electroosmotic flow (electric field) v.s. hydrodynamic flow (pressure difference)

111

112 What is Capillary Electrophoresis? (d) at low ph, Si-O Θ Si-OH cation in the double layer - at neutral ph or high ph electroosmosis > electrophoresis the net flow: anions cathode - at low ph anode anion & may never reach the detector.

113 What is Capillary Electrophoresis? Electroosmosis leads to bulk solution flow that has a flat profile across the tube because flow originates at the walls of the tubing. Hence, electro osmotic flow does not contribute significantly to band broadening the way pressure-driven flow does in liquid chromatography.

114 Although analytes migrate according to their charges within the capillary, the electroosmotic flow rate is usually sufficient to move all positive, neutral, and even negative species toward the same end of the capillary.

115 Types of capillary electrophoresis Crown ether has greater affinity for D-aa than L-aa P.524

116 Types of capillary electrophoresis (1) Capillary zone electrophoresis : cations > neutrals (unseparated) > anions (2) Micellar electrokinetic capillary electrophoresis separate neutral molecules as well as ions

117 Types of capillary electrophoresis What is a micelle? are anions & behave like a pseudostationary phase c 0 in van deemter eqn. What do you expect the van Deemter curve to look like?

118 Types of capillary electrophoresis Neutral molecules reach the detector at a time between t mc (the time for micelles to reach detector) and t 0 (absence of micelles). soluble in the micelle time inside the micelle migration time, i.e. t r : nonpolar solutes > polar

119 Homework 34-10, 34-11, 34-13, 34-16

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: