Chromatography Lecture Notes 2024-2025 PDF

TLC D R FA W Z I E L S E B A E I 1 Planner Chromatography Planar chromatographic methods include thin-layer chromatography (TLC), paper chromatography (PC), and electrochromatography. Each makes use of a flat, relatively thin layer of m...

TLC D R FA W Z I E L S E B A E I 1 Planner Chromatography Planar chromatographic methods include thin-layer chromatography (TLC), 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. Thin-layer technique is faster, has better resolution, and is more sensitive than its paper counterpart. 2 Page 1  Principles of (TLC) Thin-layer Chromatography carried out on active particulate material (silica gel or alumina) dispersed on an Inert support (flat glass plates). The mobile phase is drawn over the surface by capillary action. Advantages: Thin-layer and liquid chromatography are quite similar in regards to theory, stationary and mobile phases. Speed Low cost of the exploratory thin-layer experiments. The TLC apparatus is much simpler than an HPLC system and much less expensive to operate 3 Basic Steps of TLC Technique Preparation of the Plate Sample Application Chromatogram Development Locating of the Spots 4 Page 2  Preparation of the Plate § Slurry of the active material is uniformly spread over the plate by means of a commercially available spreader (a binder is incorporated into the slurry to enhance adhesion of the solid particles to the glass and to one another). § Air-drying overnight, or oven-drying at 80-90 °C for about 30 minutes. § Commercial plates can be conventional and high-performance plates. Conventional plates have thicker layers (200 to 250 μm) of particles with particles sizes of 20 μm or greater. High-performance plates usually have film thicknesses of 100 μm and particle diameters of 5 μm or less. 5 Sample Application § Sample application is perhaps the most critical aspect of thin-layer chromatography. § Manual application of samples is performed by touching a capillary tube containing the sample to the plate or by use of a syringe. § Mechanical dispensers, which increase the precision and accuracy of sample application, are available commercially. 6 Page 3  Chromatogram Development · § Avoid direct contact between the sample and the solvent system. The tank or chamber is preferably lined with filter paper (Plate conditioning). § As the developing solvent travels up the plate, it dissolves the sample and carries it up; the sample distributing itself between the moving solvent and the stationary phase. 7 TLC method development Solution for plate conditioning with vapor phase: twin through chambers ( + filter paper ). 8 Page 4  Horizontal Elution : Horizontal flow developing chamber in which samples are placed on both ends of the plate and developed toward the middle, thus doubling the number of samples that can be accommodated. 9 Locating of the Spots For Colored Compounds: 10 Page 5  For Colorless Compounds: Where is the spots ? We do not know. Solvent front a Rf = b/a b · Base line Iodine or sulphuric acid is used for most organic mixtures giving dark spots. Ninhydrin is used for amino acids. 2,4-Dinitrophenylhydrazine is used for aldehydes and ketones 11 Applications of TLC Technique Identification of Unknown Compounds Analysis of Reaction Mixture 12 Page 6  Applications of TLC Technique Monitoring a Chemical Reaction Determination ofthePurity ofaCompound 13 Quantitative Determination of an Unknown Concentration External Standard Method Signal Concentration Calibration curve 14 Page 7  Quantitative Analysis Calibration curve Internal Standard Method Signal ratio Concentration Internal Standard Compound 0 10 0 10 0 10 0 10 0 10 0 10 0 10 100 ng/mL 75 ng/mL 50 ng/mL 25 ng/mL 10 ng/mL 5 ng/mL Unknown 15 HPTLC Another method of detection is based on incorporating a fluorescent material into the stationary phase. The sample components quench the fluorescence of the material so that all of the plate fluoresces except where the nonfluorescing sample components are located. 16 Page 8  HPLC HIGH -PE RFO RM AN CE L I Q U I D C H R O M AT O G R A P H Y D R FA W Z I E L S E B A E I 17 High-Performance Liquid Chromatography (HPLC) What is the Instrumentation of HPLC ? How we Can Develop the HPLC Method ? What is the Applications of HPLC ? 18 Page 9  HPLC Mobile phase liquid, Stationary phase solid or liquid bonded to solid support Traditional LC is slow (no pump); flow under gravity HPLC, pressure is applied fast HPLC is used for organic and inorganic analysis 19 HPLC Advantages of HPLC ، Speed à fast ، Resolution à high ، Accuracy à error < 1% ، Sensitivity à detection limit 10-10g ، Automation à possible (major advantage in industry) Limitations of HPLC ، Expensive instrumentation ، Experience required ، Universal detectors à not sensitive ، Sensitive detectors à not universal 20 Page 10  Instrumentation of HPLC 21 22 Page 11  injection Solvent valve mixing Mobile-Phase Reservoir valve Pump Chart Detector Recorder Mobile phase reservoir Waste Often the reservoirs contain a filtration system for filtering dust and particulate matters from the solvent to prevent these particles from damaging the pumps or injection valves or blocking the column. The reservoirs are equipped with a degasser for removing dissolved gases- usually oxygen and nitrogen-that interfere by forming bubbles in the column and the detector. 23 injection Solvent valve mixing valve Pump HPLC Pump Chart Detector Recorder Mobile phase The Function: reservoir Waste The pump provide a flow of the mobile-phase through the HPLC injector, column, and detector. The requirements of standard HPLC pump include: § Generation of pressures up to 6000 Ibs/in2. § Pulse-free output. § Flow rate ranging from 0.1 to 10 ml/min. § Made of corrosion-resistant materials (stainless steel). 24 Page 12  Types of HPLC Pump Types: A: According to pressure: ، Constant pressure - Constant flow ، Low pressure - high pressure B: According to flow: ، Fixed - variable C: According to elution: ، Isocratic -gradient 25 HPLC Pump Constant-Pressure Pump: Advantages: Simple, inexpensive, easy to operate, and free from pulsations, resulting in smooth baselines. Disadvantages: Flow rate must be monitored carefully and constantly. Causes: change in the solvent viscosity (due to change in the temperature or composition) Effect: influence both qualitative and quantitative analysis. How: change in the flow rate reflects on change in retention volume (used for matching in the qualitative analysis). This type is used only for column packing 26 Page 13  Constant-Flow Pump: Advantages: Ability to repeat elution volume and peak area (regardless of viscosity changes or column blockage, up to the pressure limit of the pump). This type is the most widely used in all common HPLC applications 27 Mobile Phase Must do the following: ، solvate the analyte molecules and the solvent they are in ، be suitable for the analyte to transfer “back and forth” between during the separation process Must be: ، Compatible with the instrument (pumps, seals, fittings, detector, etc) ، Readily available (often use liters/day) ، Compatible with the stationary phase ، of adequate purity ÷ spectroscopic and trace-composition usually! ، Not too compressible (causes pump/flow problems) ، Filtered, and free of gases 28 Page 14  Isocratic versus Gradient Elution Isocratic elution has a constant mobile phase composition ، Can often use one pump! ، Mix solvents together ahead of time! ، Simpler, no mixing chamber required ، Limited flexibility, not used much in research ÷ mostly process chemistry or routine analysis. Gradient elution has a varying mobile phase composition ، Uses multiple pumps whose output is mixed together ÷ often 2-4 pumps (binary to quaternary systems) ، Changing mobile phase components changes the polarity index ÷ can be used to subsequently elute compounds that were obviously different in their capacity values. ÷ Column has to re-equilibrate to original conditions after each run (takes additional time). 29 30 Page 15 31 Typical HPLC Pump (runs to 4,000+ psi) Polymer Piston, often made of industrial sapphire or ruby 32 Page 16  Polarity Index for Mobile Phases….. The polarity index is a measure of the relative polarity of a solvent. It is used for identifying suitable mobile phase solvents. The more polar your solvent is, the higher the index. You want to try to choose a polarity index for your solvent (or solvent mixture) that optimizes the separation of analytes ÷ Usually the index is a starting point ÷ The polarity of any mixture of solvents to make a mobile phase can be modeled to give a theoretical chromatogram ÷ Usually, optimization of solvent composition is experimental 33 A useful guide when using the polarity index is that a change in its value of 2 units corresponds to an approximate tenfold change in a solute’s capacity factor. EXAMPLE A reverse-phase HPLC separation is carried out using a mobile-phase mixture of 60% v/v water and 40% v/v methanol. What is the mobile phase’s polarity index? If the capacity factor was 22 for a mobile phase of only water, what will be the capacity factor after shifting to the new mobile phase mixture SOLUTION From Table, we find that the polarity index is 10.2 for water and 5.1 for methanol. PAB′ = (0.60)(10.2) + (0.40)(5.1) = 8.2 Polarity index change= 10.2 – 8.2 =2 New capacity factor = 22/10 = 2.2 34 Page 17 35 36 Page 18  Injection in HPLC Usually 5 to 1000 mL volumes, all directly onto the column Injector is the last component before the column(s) A source of poor precision in HPLC ، errors of 2-3 %RSD are due just to injection ، other errors are added to this ، due to capillary action and the small dimensions/cavities inside the injector Types: ، Septum - 6-PORT Rotary Valve - Automatic injectors Rotary Valve is the standard manual injector ، Two positions, load and inject in the typical injector ، Injection loop internal volume determines injection volume. 37 injection Solvent valve Sample Injection System mixing valve Pump Chart Detector Recorder Mobile phase reservoir Waste The Function: Introduction of the samples into the HPLC system with high precision, without interruption of the mobile phase flow. Injection valve (loop) is preferred for quantitative analysis 38 Page 19  LOAD (the sample loop) Inject (move the sample loop into the mobile phase flow) 39 Columns and Stationary Phases b HPLC is largely the domain of packed columns b Columns are made from stainless steel b Columns: length, 3-50 cm; diameter 3 – 10 mm b Packed with stationary phase : 1 to 20 mm diameter b Particles: Pellicular glass with a thin layer of porous material (10 50 mm ) porous (such as silica gel), 1 -10 mm Porous silica particles gives better column efficiency, sample capacity, and speed of analysis b --microbore/capillary columns (open tubular column) 40 Page 20  Columns and Stationary Phases…... HPLC is largely the domain of packed columns ، some research into microbore/capillary columns is going on. ، Molecules move too slowly to be able to reach and therefore “spend time in” the stationary phase of an open tubular column in HPLC. Stationary phases are particles which are usually about 1 to 20 mm in average diameter (often irregularly shaped) ، In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil). ، In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin) 41 van-Deemeter plot 42 Page 21  Stationary Phases Polar (“Normal Phase): ، Silica, alumina ، Cyano, amino or diol terminations on the bonded phase Non-Polar (“Reversed Phase”) ، C18 to about C8 terminations on the bonded phase ، Phenyl and cyano terminations on the bonded phase Mixtures of functional groups can be used!! Packed particles in a column require: ، Frits at the ends of the column to keep the particles in ، Filtering of samples to prevent clogging with debris ، High pressure pumps and check-valves ، Often a “Guard Column” to protect the analytical column 43 injection valve HPLC Detector Solvent mixing valve Pump The function: Detector Recorder Chart Monitoring the mobile phase Mobile phase reservoir Waste as it emerges from the column.. The ideal characteristics: 1. Adequate sensitivity for the particular task. 2. Good stability and reproducibility. 3. Wide linear dynamic range of response. 4. Short response time that is independent on flow rate. 5. Insensitive to changes in solvent, flow rate, and temperature. 6. Cell design that eliminates remixing of the separated bands. 7. High reliability and ease of use. 8. Non-destructive for the sample. 9. Ease of operation 44 Page 22  HPLC Detector Types of HPLC Detectors: A. Bulk Property Detectors. B. Solute Property Detectors § Bulk Property Detectors. : Respond to some physical property of the mobile-phase (refractive index, dielectric constant, or density). Advantages: they are universal in application. Disadvantages: they have poor sensitivity and limited range 45 HPLC Detector § Solute Property Detectors. They respond to some physical or chemical property of solutes (UV absorbance, fluorescence, or diffusion current). Advantages: They high sensitivity and a wide range. Disadvantages: They are more selective; more than one detector may be required to meet the demands of an analytical problem. 46 Page 23  HPLC Detector Characteristics of Typical HPLC Detectors : Type Response Sensitivity (ng/mL) Refractive index Universal 1000 Conductimetric Selective 100 UV/visible absorption Selective 10 Mass-spectrometry Selective 0.1 Fluorescence Selective 0.001 47 48 Page 24 49 Standard Absorbance Detector…. Single Beam UV-VIS instrument with a flow-through cell (cuvette) Can use any UV-VIS with a special flow cell ، Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit. Usually utilize Hg vapor lamps and 254 nm line (generic) ، Can be set to other wavelengths (most) ، Simple filter detectors no longer widely used Non-destructive, not-universal ، not all compounds absorb light ، can pass sample through several cells at several different wavelengths. Usually zeroed at the start of each run using an electronic software command. 50 Page 25  Diode Array Detector (DAD) The more common tool for research-grade HPLC instruments ، quite versatile... Advances in computer technology since ~1985 or so have lead to the development of Diode Array instruments Non-destructive, non-universal DAD scans a range of wavelengths every second or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum! ، Huge volumes of data ، Detailed spectra for each peak and each region of each peak 51 52 Page 26  Refractive Index Detector One of a very few Universal HPLC detectors. Non-destructive Responds to analytes changing the RI of the mobile phase ، requires a separate reference flow of mobile phase Extremely temperature sensitive, usually heated ، sensitive to temp changes of +/- 0.001 °C No longer really widely used ، Absorbance detectors are relatively cheap. Useful for process work, on-line monitoring, etc. 53 ELSD (Evaporative Light Scattering Detector) o Universal, destructive o Useful for very large molecules, and a wide linear range o Analytes are de-solvated in the detector o Molecules pass through what is essentially a large cuvette for a UV-VIS instrument o The reduction in light intensity detected (due to scattering by the analytes) is measured o The larger and more concentrated a particular molecule is, the greater the scattering. 54 Page 27 55 Other Detectors... Mass Spec ، problem is interfacing a liquid flow with a vacuum system! Electrochemical Conductivity Fluorescence 56 Page 28  injection valve HPLC Recorder Solvent mixing valve Pump Chart Detector Recorder Mobile phase reservoir Waste Sample injected Peaks correspond to individual components 57 Common HPLC Techniques q Partition chromatography Normal phase chromatography Reverse phase chromatography q Adsorption chromatography q Ion chromatography q Size exclusion chromatography q Ion-pair chromatography q HILIC chromatography q affinity chromatography q Chiral chromatography 58 Page 29  Types of chromatography 59 Partition Chromatography Normal phase chromatography: ، Stationary phase : polar (e.g. thin layer of H2O surrounding silica gel or alumina solid support ) ، Mobile phase : non-polar solvents (hexane, chloroform) Reverse phase chromatography: ، Stationary phase : non-polar (C-18, C-8, CN, phenyl bonded to solid support like silica gel) ، Mobile phase : polar solvents (water + MOH, CH3CN) ، ↑ non polarity of the mobile phase ↑ its strength ↓ retention time. (Water > MOH > CH3CN in polarity) 60 Page 30  Partition Chromatography The most widely used type of HPLC is partition chromatography in which the stationary phase is a second liquid that is immiscible with the liquid mobile phase. Separation is achieved according to difference in partition power (partition coefficient (K)) of different analytes. Partition chromatography can be subdivided into liquid-liquid and liquid- bonded-phase chromatography. In liquid-liquid partition chromatography, the stationary phase is a solvent held in place by adsorption of the surface of the packing particles (e.g. silica surrounded by a thin film of H2O (a type of normal phase chromatography)) In liquid-bonded-phase chromatography, the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds. The bonded-phase method predominate because of their greater stability and compatibility with gradient elution. 61 Partition Chromatography Most bonded-phase packings are prepared by reaction of an organochlorosilane with the -OH groups formed on the surface of silica particles Silica surface bonded-phase packing In reversed phase chromatography, the organic group R is often a straight chain octyl- (C8) or octyldecyl- (C18) group. Other organic functional groups include aliphatic amines, ethers, and nitriles to obtain a wide range of different polarity stationary phases 62 Page 31 RPLC-Partitioning Alkyl Hydrophobic interactions chains from C1 to C30 Polymers: PS-DVB H H Si H Si H Si H O O O O O O O O Si O Si O Si O Si O Si O Si O Si O Si O C8 C18 PHENYL 63 RPLC-Partitioning OctaDecyl-Siloxane phases (ODS or C18) A great variety of stationary phases None of them is identical to the other!! 64 Page 32 It has been estimated that more than three-quarters of all HPLC separations are currently performed with reversed-phase, bonded, octyl- or octyldecyl siloxane packings. With such preparations, the long-chain hydrocarbon groups are aligned parallel to one another and perpendicular to the surface of the particle, giving a brushlike, nonpolar hydrocarbon surface. The mobile phase used with these packings is often an aqueous solution containing various concentrations of such solvents as methanol, acetonitrile, or tetrahydrofuran. 65 NPLC-Partitioning Acidic Polar interactions HO HO H Basic Dipole N≡C O N H H H Si H Si H Si H O O O O O O O O Si O Si O Si O Si O Si O Si O Si O Si O CYANO DIOL AMINO 66 Page 33  HPLC Columns 67 Adsorption Chromatography Analyte is adsorbed to the surface of a polar stationary phase and separation is done according to difference in adsorption power between different analytes. Analyte will compete with the mobile phase to occupy sites on the surface of the stationary phase. Retention times become longer as the polarity of the analyte increases Silica gel or alumina (Al2O3) are usually used as stationary phases. MP is non-polar (hexane or heptane + CHCl3, or ethyl acetate, …….) Used for the separation of non-polar (water-insoluble) compounds Also used for separation of certain isomers 68 Page 34  Ion Exchange Chromatography Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. It can be used for almost any kind of charged molecule including large anions, cations, proteins, small nucleotides and amino acids. It is often used in pharmaceutical analysis, protein purification, water analysis. Ion exchange chromatography retains analyte molecules based on ionic interactions (charge size). Polyvalent ions are more strongly held by ion exchange resins than are univalent ions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into: 1.cation exchange chromatography 2.anion exchange chromatography. 69 Ion exchangers – Functional groups Cation exchanger Carboxymethyl (R-CH2COO-); phosphor (R-PO3-); Sulphopropyl (R-C3H6SO3 )- Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group : R-X- C+ + M+B- R-X- M+ + C++ B- 70 Page 35  Ion exchangers – Functional groups Anion exchanger Aminoethyl (R-C2H2NH2) Diethylaminoethyl (R-C2H2 N(C2H2)2) Quaternary aminoethyl (R4N+) Anion exchange chromatography retains anions using positively charged functional group: R-X+ A- + M+B- R-X+ B- + M+ + A- 71 Ion Exchange Chromatography The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions or lowering the pH The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance. 72 Page 36  Ion Exchange Chromatography 73 Size-exclusion Chromatography Also known as molecular exclusion chromatography, gel permeation or gel filtration. Separation is based on the molecular size of the components. The liquid phase passes through a porous gel which separates the molecules according to its size (shape and hydration). Molecules that are too large for the pores of the porous packing material on the column elute first, small molecules that enter the pores elute last. Elution rates of the rest depend on their relative sizes 74 Page 37  Size-exclusion Chromatography Numerous size-exclusion packings are on the market. Some are hydrophilic for use with aqueous mobile phases; others are hydrophobic and are used with nonpolar organic solvents. Chromatography based on the hydrophilic packings is sometimes call gel filtration, while that based on hydrophobic packings is termed gel permeation. With both types of packings, many pore diameters are available. 75 Types of chromatography 76 Page 38 77 Ion-pair Chromatography Ion-pair chromatography is a type of chromatography in which easily ionizable species are separated on reversed-phase columns. In this type of chromatography, a large organic counter ion is added to the mobile phase as an ion-pairing reagent. For acidic compounds (carry –ve charge), we add to the mobile phase a positively charged counter ion such as tetrabutyl ammonium bromide. For basic compounds (carry +ve charge), we add to the mobile phase a negatively charged counter ion such as hexane sulfonate R-SO3-. Separation is affected by pH, ionic strength of buffers, temperature, concentration and type of organic co-solvent(s). Used mainly for ionic (acidic or basic) drugs. 78 Page 39 79 HILIC chromatography: Same stationary phases as in normal phase chromatography Same eluents (MeCN/water) as in reversed phase chromatography Mechanism is explained via liquid-liquid partitioning Stationary phase is covered with water layer, while the mobile phase contains less water Analyte partitions between the water layer and the mobile phase Polar compounds show higher retention and elute later 19 80 Page 40 81 Some Advantages HILIC has many specific advantages over conventional NP-LC and RP-LC. For example, it is suitable for analyzing compounds in complex systems that always elute near the void in reserved-phase chromatography. Polar samples always show good solubility in the aqueous mobile phase used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in NP-LC. Expensive ion pair reagents are not required in HILIC, and it can be conveniently coupled to mass spectrometry (MS), especially in the electrospray ionization (ESI) mode. In contrast to RP-LC, gradient elution HILIC begins with a low-polarity organic solvent and elutes polar analytes by increasing the polar aqueous content 82 Page 41 83 Affinity Chromatography Affinity chromatography employs a ligand (biologically active molecule bonded covalently to the solid matrix) which interacts with its homologous antigen (analyte) as a reversible complex that can be eluted by changing buffer conditions. Typical affinity ligands are antibodies, enzymes, or other molecules that reversibly and selectively bind to analyte molecules in the sample. Other molecules have no retention and just cleaned throughout the column. Affinity chromatography has the major advantage of extraordinary specificity. The primary use is in the rapid isolation of biomolecules during preparative work.. 84 Page 42  Types of chromatography 85 Chiral Chromatography Used for separation of the enantiomers. Separation is carried out on special chiral stationary phases. Can also be achieved on regular stationary phases by formation of diastereomers via derivatizing agents (chiral mobile phase additives). Preferential complexation between the chiral resolving agent (additive or stationary phase) and one of the isomers results in a separation of the enantiomers. The chiral resolving agent must have chiral character itself in order to recognize the chiral nature of the solute. When used as an impurity test method, the sensitivity is enhanced if the enantiomeric impurity elutes before the enantiomeric drug. 86 Page 43 87 Choice of HPLC Technique Separation is affected by: ، Components must be retarded on a phase (retention), ، Components must be retarded differently (selection) ، The peaks of the components must be sharp to avoid overlapping (efficiency) 88 Page 44  Choice of HPLC Technique Stationary phase is selected according to: 'like dissolves like„ Look for a particular group in the molecule of the solute and find the best match of the stationary phase Solutes MW > 10,000 à use size-exclusion chromatography Low MW ionic species à ion-exchange chromatography Small nonionic species à partition methods A prediction of the correct chromatographic system cannot be made with certainty, however, and must be confirmed by experiment. 89 What does the analyst do? q Select the correct type of separation for the analyte(s) of interest, based on the sample type (among other factors). q Select an appropriate column (stationary phase) and mobile phase q Select an appropriate detector based on whether universal or compound-specific detection is required or available q Optimize the separation using standard mixtures q Analyze the sample 90 Page 45  Which technique will be appropriate? Applications of liquidchromatography. Methods can be chosen based on solubility and molecular mass. In many cases, for small molecules, reversed-phase methods are appropriate. Techniques toward the bottom of the diagram arebest suited for high molecular mass (M. 2000). 91 How we Can Develop the HPLC Method ? 92 Page 46  Optimization of Separations in HPLC Analytemobile phase « Analyte stationary phase Correct choice of column so the above equilibrium has some meaningful (non-infinity, non-zero) K. Correct choice of mobile phase Decision on the type of mobile phase composition ،constant composition = isocratic ،varying composition = gradient elution Determination if flow rate should be constant or be varied ،usually it is constant Decision on heating the column or not ،usually we do NOT heat HPLC columns…. 93 What is the Applications of HPLC ? Peaks correspond to individual components Qualitative Analysis Separation of Mixture Components Compound Impurity Purification of Compounds Authentic Unknown Identification of Compounds 94 Page 47  Quantitative Analysis Calibration curve External Standard Method Peak area Concentration 0 10 0 10 0 10 0 10 0 10 0 10 0 10 100 g/mL 75 g/mL 50 g/mL 25 g/mL 10 g/mL 5 g/mL Unknown 95 Calibration curve Quantitative Analysis Peak area ratio Internal Standard Method Concentration Internal Standard Compound 0 10 0 10 0 10 0 10 0 10 0 10 0 10 100 g/mL 75 g/mL 50 g/mL 25 g/mL 10 g/mL 5 g/mL Unknown 96 Page 48  GC D R FA W Z I E L S E B A E I 97 Introduction GC is based upon partition of solutes between the stationary phase and an inert carrier gas. the mobile phase does not interact with molecules of the analyte. The only function of the mobile phase is to transport the analyte through the column.. Sample is vaporized before entering the column. § GC is classified into: §Gas-solid chromatography (adsorption) §Gas-liquid chromatography (partition) 98 Page 49  Type of Samples Any compound that can be volatilized: Molecular weight ranges from 2 to 1000 (samples having high m. wt. e.g. polymers can not be used in direct GC). The sample may be organic or inorganic (GC can not separate ionic compounds). Compounds should be thermally stable: Amino acids, sugars, and proteins decompose at high temperatures and can not be analyzed. 99 Advantages and Limitations Advantages: § High resolution (many components in a given- sample can be identified and quantitatively determined ). § High speed (short time of analysis). § High sensitivity (10-9 -10-12 gm).. § High accuracy. Limitations: § Samples must be volatile and thermally stable below ~ 400°C. § The most commonly used detector (Flame ionization detector) is destructive (samples are decomposed and can not be collected) § Due to its high sensitivity, dirty samples such as blood or tissues require intensive clean up. § GC cannot identify the compound surely and another instrument such as IR or mass spectrometer must be used for complete compound identity. § Some training and experience are necessary. 100 Page 50  GC Theory q An inert gas such as helium is passed through the column as a carrier gas and is the moving phase. q A sample is injected into a port which is much hotter than the column and is vaporized. q The gaseous sample mixes with the helium gas and begins to travel with the carrier gas through the column. q As the different compounds in the sample have varying affinity to the column liquid stationary phase and as these compounds cool a bit, they are deposited in the stationary phase. However, the column is still hot enough to vaporize the compounds and they will do so but at different rates since they have different boiling points. q The process is repeated many, many times along the column. Eventually the components of the injected sample are separated and come off of the column at different times (called "retention times"). 2-101 GC Theory q There is a detector at the end of the column which signals the change in the nature of the gas flowing out of the column. q Recall that helium is the carrier gas and will have a specific thermal conductivity, for example. Other compounds have their own thermal conductivities. q The elution of a compound other than helium will cause a change in conductivity and that change is converted to an electrical signal. q The detector, in turn, sends a signal to a strip chart recorder or to a computer. Detectors come in several varieties, for example, thermal detectors, flame-ionization and electron capture detectors. 2-102 Page 51  GC Theory q The chromatogram shows the order of elution (order of components coming off the column), the time of elution (retention time), and the relative amounts of the components in the mixture. q The order of elution is related to the boiling points and polarities of the substances in the mixture. q In general, they elute in order of increasing boiling point but occasionally the relative polarity of a compound will cause it to elute "out of order". 2-103 GC Theory The observed elution pattern appears below. Notice the reversed elution of toluene and 4-methyl-2-pentanone. 2-104 Page 52  Instrumentation Flow meter Injector Septum Detector GC Pressure Flow Chart regulator controller Recorder Oven Gas supply Column 105 Pressure FlowFlow meter Gas Supply Flow regulator controller Injector controller Septum Detector GC Pressure chart regulator Recorder Factors affecting the Oven choice of carrier gas Gas Column Gas supply supply 1. Viscosity: § affects pressure applied, which affect flow rate. 2. Reactivity: § Carrier gases should be sufficiently inert. § They must neither cause damage to the stationary phase nor to the sample components. § Caution should be used with H2. Even though hydrogen offers many advantages as a carrier gas, it is less inert than the other gases. 3. Safety: § Mixtures of 4% of H2 in air for instant, is explosive. Nothing can go wrong as long as hydrogen is in a leakage-free gas system. 106 Page 53  Factors affecting the choice of carrier gas 4. Carrier gas and detection method: q The type of carrier gas has little effect on the most widely used detector, the flame ionization detector (FID). N2, He, and H2 can be used. H2 has the advantage that it can be employed as carrier gas and as fuel gas FID also needs oxygen or air, in addition to fuel for the combustion. q The most universal detector, the thermal conductivity detector (TCD), works best with H2 or He. They have a much greater thermal conductivity compared to N 2. Therefore, organic compounds can be determined with much better sensitivity. q The electron capture detector (ECD) requires an argon- methane mixture. 107 Factors affecting the choice of carrier gas 5. Purity: q 99.999% or more pure gas should be used q Impurities: moisture, air (O2), gaseous hydrocarbons can interact with the sample and may cause column deterioration or affect the detector performance. q Therefore, 3 filters are usually used: § a moisture filter to remove water and, sometimes traces of oil from connections or pressure gauge. § an oxygen filter to remove oxygen and traces of sulfur and chlorine compounds. § for FID-fuel gases: an active charcoal filter to remove organic compounds. q The filters in GC should be Installed in a particular order: first, the moisture filter and then the oxygen filter. The stationary phase can be destroyed by oxygen which was dissolved in the water and was released after filtering. “Sometimes the filters are placed in reverse order“ 108 Page 54  Flow meter Flow meter Flow Sample Injection System controller Pressure Injector Septum Injector Septum Detector Detector GC chart regulator Recorder Recorder Carrier gas Oven Preparation of the Sample : Oven Gas Column Column supply q Samples in GC must be volatile. Samples which are non volatile are converted into a volatile derivative. § The most common derivatization method is silylation (reaction of trimethylsilyl, - Si(CH3), with an active hydrogen atom in the analyte (carboxylic acids, amines, imines, alcohols, phenols, and thiols). § Inorganic metals (aluminum, beryllium, and chromium) can be analyzed by GC via formation of stable, volatile metal chelates via reaction with trifluoroacetylacetone (TFA) and hexafluoroacetylacetone (HFA). 109 Derivation of Glucose with Trimethylchlorosilane (Silylation) 110 Page 55  Sample Injection System Injection of the Sample : q The injector block; is heated to a temperature that is at least 50 °C above the sample component with the highest boiling point; this to ensure rapid vaporization of the entire sample. q The injection system is a self-sealing silicone septum. q The sample is injected by a microliter syringe. The needle of the syringe is inserted through the septum and the sample is injected smoothly into the heated metal block at the head of the column. 111 Sample Injection System Injection techniques : q Capillary columns require samples that are smaller by a factor of 100 or more than packed columns. For these columns, a sample splitter is often needed to deliver a small known fraction (1:100 to 1:500) of the injected sample, with the remainder going to waste. Commercial gas chromatographs intended for use with capillary columns incorporate such splitters, and they also allow for splitless injection when packed columns are used. q For the most reproducible sample injection, newer gas chromatographs use autoinjectors and autosamplers. Standard deviations as low as 0.3% are common with autoinjection systems. 112 Page 56  Sample Injection System Injection techniques : q On-column injector (100 % of the sample is introduced to column) q Split-splitless injector (0.5 – 5 % of the sample is introduced to column) q Autoinjectors. Injection volume : q For gases: 10 – 50 µl q For liquids: 0.1 – 10 µl 113 GC column The GC column contains the stationary phase on which actual separation of sample components is affected. There are two types of GC columns: q Packed column: is constructed from glass, stainless steel, copper, or aluminum and filled with a particulate solid support, mostly a glass beads, or fluorocarbon polymers on which a thin film of liquid stationary phase is retained. q Capillary column: the stationary phase was a film of liquid a few tenths of a micrometer thick that uniformly coated the interior of a capillary tubing. In the late 1950s, such open tubular columns were constructed, with performance characteristics of 300,000 plates or more being described. However, the most significant development in capillary GC occurred in 1979 when fused-silica capillaries were introduced. Capillary columns are now mostly used. 114 Page 57  GC column q Columns vary in length from less than 2 m to 50 m or more. In order to fit into the column oven, they are usually formed as coils. q The control of column temperature is critical to attain a good separation and better reproducibility in GC, thus the column is located inside a thermostated oven to control the temperature. (Temperature programming and gradient elution) q Types of capillary columns: ، Support-coated open tubular (SCOT) columns ، Wall-coated open tubular (WCOT) ، Fused-silica open tubular (FSOT); 115 GC column q In support-coated open tubular columns, the inner surface of the capillary is lined with a thin film (30 μm) of a solid support material, such as diatomaceous earth, on which the liquid stationary phase is adsorbed. Generally, the efficiency of a SCOT column is less than that of a WCOT column but significantly greater than that of a packed column. q Wall-coated columns are capillary tubes coated with a thin layer of the liquid stationary phase. Early WCOT columns were constructed of stainless steel, aluminum, copper, or plastic. Subsequently, glass was used. Often, an alkali or borosilicate glass was leached then subsequent etching roughened the surface, which bonded the stationary phase more tightly. 116 Page 58  GC column q Fused-silica capillaries are drawn from specially purified silica that contain minimal amounts of metal oxides. These capillaries have much thinner walls than their glass counterparts. They are given added strength by an outside protective polyimide coating. The resulting columns are quite flexible and can be bent into coils. q Commercial fused silica columns offer several important advantages over glass columns, such as physical strength, much lower reactivity toward sample components, and flexibility. For most applications, they have replaced the older type WCOT glass columns. q Fused-silica columns with inside diameters of 0.32 and 0.25 mm are very popular. Higher-resolution columns are also sold with diameters of 0.20 and 0.15 mm. Such columns are more troublesome to use and are more demanding on the injection and detection systems. Thus, a sample splitter must be used to reduce the size of the sample injected onto the column, and a more sensitive detector system with a rapid response time is required 117 GC column 118 Page 59  Stationary Phases Requirements: 1. low volatility (B.P. > 100°C higher than the maximum operating temperature for the column); 2. thermal stability; 3. chemical inertness; 4. solvent characteristics such that k' and α values for the solutes to be resolved fall within a suitable range. The principle of “like dissolves like” is applied 119 Stationary Phases The following table lists the most widely used stationary phases for both packed and open tubular column gas chromatography that can probably provide satisfactory separations for 90% or more of samples. Five of the stationary phases are based on polydimethyl siloxanes either per se or substituted with different percentages of other groups for their methyl groups. The fifth is a polyethylene glycol that finds widespread use for separating polar species. 120 Page 60  Most Common Stationary Phases Stationary phases are in order of increasing polarity. 5% phenyl polydimethyl siloxane has a phenyl ring bonded to 5% (by number) of the silicon atoms in the polymer. 121 Stationary Phases Bonded and Cross-Linked Stationary Phases: With use, untreated columns slowly lose their stationary phase due to “bleeding” in which a small amount of immobilized liquid is carried out of the column during the elution process. Bleeding is exaggerated when a column must be rinsed with a solvent to remove contaminants. The purpose of bonding and cross-linking is to provide a longer lasting stationary phase that can be rinsed with a solvent when the film becomes contaminated. Bonding consists of attaching a monomolecular layer of the stationary phase to the silica surface of the column by a chemical reaction. 122 Page 61  Stationary Phases Cross-linking is accomplished in situ after a column is coated with the stationary phase. For example by incorporating a peroxide into the original liquid. When the film is heated, reaction between the methyl groups in the polymer chains is initiated by a free radical mechanism. The polymer molecules are then cross-linked through carbon-to-carbon bonds. Film Thickness Commercial columns are available having stationary phases that vary in thickness from 0.1 to 5 μm. Thick films are used with highly volatile analytes because such films retain solutes for a longer time, thus providing a greater time for separation to take place. For most applications with 0.25 or 0.32 mm columns, a film thickness of 0.25 μm is recommended. 123 Programmed Temperature Gas Chromatography Column temperature is an important variable that must be controlled to a few tenths of a degree for precise work. Thus, the column is normally housed in a thermostated oven. The optimum column temperature depends on the boiling point of the sample and the degree of separation required. Roughly, a temperature equal to or slightly above the average boiling point of a sample results in a reasonable elution time (2 to 30 min). For samples with a broad boiling range, it is often desirable to use temperature programming whereby the column temperature is increased either continuously or in steps as the separation proceeds. Shorter elution times can be obtained using higher temperature on the expense of attained resolution and capacity. 124 Page 62  Programmed Temperature Gas Chromatography Effect of temperature on gas chromatograms. (a) Isothermal at 45 C. (b) Isothermal at 145 C. (c) Programmed at 30 to 180 C. 125 Flow meter Flow GC detector controller Pressure Injector Septum Detector GC chart regulator Recorder The function: Oven Monitoring the carrier gas as it emerges from Gas Column supply the column. The requirements of an ideal GC detector: The same as HPLC detectors. Types of GC detectors: § Thermal conductivity detector (TCD) § Electron capture detector (ECD) § Flame ionization detector (FID) § Nitrogen phosphorous detectors (NPD) 126 Page 63  Flame Ionization Detector (FID) n Principle: High temperature of hydrogen flame (H2/O2 or H2/air) ionizes compounds eluted from column into flame. The ions collected on collector electrode and the produced current is recorded. n It is destructive detector. n Used for flammable (organic) compounds n Its good sensitivity and wide linear range make it more versatile over other detectors. 127 Schematic Diagram of Flame Ionization Detector 128 Page 64  Thermal Conductivity Detector (TCD) q Principle: Measures the changes of thermal conductivity due to the sample (mg). q Universal non-destructive detector. q Being universal as well as its simplicity, stability and low cost are the most prominent advantages of TCD. Note: The TCD will respond to any substance different from the carrier gas as long as its concentration is sufficiently high enough. Sample can be recovered. 129 Thermal Conductivity Detector (TCD) Principle: The ability of a gas to dissipate heat, i.e. its thermal conductivity, from a heated body shall change with the composition of the gas. Composition: Consists of two cells, made within a metal block, termed as reference cell and sample cell. Each cell has a resistance wire or filament that possesses a high temperature coefficient or resistance i.e., the resistance varies appreciably with slight variation in temperature. These two filaments are included in two arms of a Wheatstone Bridge. When the sample components are eluted, the thermal conductivity of the column effluent decreases leading to an increase in the temperature of the sample filament. The Wheatstone Bridge gets unbalanced. The off balance current is transmitted to the recorder. 130 Page 65  Schematic Diagram of thermal conductivity Detector 131 Relative Thermal Conductivity Relative Thermal Compound Conductivity Carbon Tetrachloride 0.05 Benzene 0.11 Hexane 0.12 Argon 0.12 Methanol 0.13 Nitrogen 0.17 Helium 1.00 Hydrogen 1.28 132 Page 66  Electron Capture Detector n Solute specific detector. Used for electrophilic compounds containing halogens (Fl, Cl, Br, I) e.g. pesticide analysis (picogram), Insecticides, vinyl chloride, and fluorocarbons. n It is the most sensitive as well as selective detector. It need a lot of skill to achieve dependable results. 133 Electron Capture Detector Principle: Electrophilic compounds will decrease the electron current generated from the carrier gas using b-particles emitter (radio active source). Composition: b-Particles emitter (3H or 63Ni) is used to liberate free electrons from the carrier gas molecules that is conducted to an anode giving a baseline signal. Ionization : N2 (Nitrogen carrier gas) + b (e) = 2 N+ + 2e When an electrophilic compound is being eluted from the column, it withdraws electrons and decrease the signal giving an elution peak. X (F, Cl and Br) containing sample + e à X- (heavy negative ions are less mobile negative signal) Ion recombination : 2 X- + 2 N+ = 2 X + N2 134 Page 67  Schematic Diagram of Electron Capture Detector 135 MS detector One of the most powerful detectors for GC is the mass spectrometer. The combination of gas chromatography and mass spectrometry is known as GC/MS. A mass spectrometer measures the mass-to-charge ratio (m/z) of ions that have been produced from the sample. Most of the ions produced are singly charged (z = 1) so that mass spectrometrists often speak of measuring the mass of ions when mass-to- charge ratio is actually measured. GC/MS instruments have been used for the identification of thousands of components that are present in natural and biological systems. With mass spectrometry, we can not only determine that a peak is due to more than one component, but we can also identify the various unresolved species. GC has also been coupled with tandem mass spectrometers and with Fourier transform mass spectrometers to give GC/MS/MS or GC/MSn systems, which are very powerful tools for identifying components in mixtures. 136 Page 68  MS detector Typical outputs for a GC/MS system. In (a) the total ion chromatogram of an extract from a termite sample is shown. In (b) the ion at m/z = 168 was monitored during the chromatogram. In (c), the complete mass spectrum of the compound eluting at t = 10.46 minutes is presented, allowing it to be identified as b-carboline norharmane, an important alkaloid. 137 GC detector 138 Page 69  GC Recorder Sample injected Peaks correspond to individual components 139 Applications of GC ? Peaks correspond to individual components Qualitative Analysis Separation of Mixture Components: Authentic Identification of Compounds: Unknown Retention time comparison FT-IR and MS detectors The application of the technique to the qualitative analysis of complex samples of unknown composition is limited because tR may not be enough. This limitation has been largely overcome by linking chromatographic columns directly with ultraviolet, infrared, and mass spectrometers to produce hyphenated instruments 140 Page 70  Applications of GC ? Peaks correspond to individual components Qualitative Analysis Pyrolysis gas chromatography It is used for the identification of non-volatile materials (plastics, natural and synthetic polymers, and some microbiological materials. It is based on the fingerprint chromatogram for the sample, which results from its thermal dissociation and fragmentation. 141 Quantitative Analysis Calibration curve External Standard Method Peak area Concentration 0 10 0 10 0 10 0 10 0 10 0 10 0 10 100 ng/mL 75 ng/mL 50 ng/mL 25 ng/mL 10 ng/mL 5 ng/mL Unknown 142 Page 71  Quantitative Analysis Calibration curve Peak area ratio Internal Standard Method Concentration Internal Standard Compound 0 10 0 10 0 10 0 10 0 10 0 10 0 10 100 ng/mL 75 ng/mL 50 ng/mL 25 ng/mL 10 ng/mL 5 ng/mL Unknown 143 Aspects of GC Applications Food Analysis Aromatic compounds such as fatty acids, esters, aldehydes, terpenes, etc present in food, volatile oils, perfumes and beverages can be easily analyzed using GC. GC can also be used to detect spoilage or possible contamination of food. Colorants, preservatives and other food additives can also be monitored using GC-MS. Drug Analysis GC is widely applied to identification of the active components, possible impurities as well as the metabolites. In pharmaceutical industry, GC-MS is used in research and development, production, and quality control. 144 Page 72 Environmental Analysis One of the most important environmental applications of gas chromatography is for the analysis of numerous organic pollutants in air, water, and wastewater. e.g. Chlorinated pesticides, dichlorodiphenyltrichloro- ethane (DDT), permethrin… etc are present in the environment at very low concentrations and are found among many of other compounds. GC, with its high sensitivity and high separating power, is mostly used in the analysis of environmental samples. Forensic Analysis In forensic cases, very little sample is available, and the concentration of the sample components may be very low. GC is a useful due to its high sensitivity and separation efficiency. Petroleum Industry Gas chromatography is ideally suited for the analysis of petroleum products, including gasoline, diesel fuel, and oil. 145 Typical chromatogram for the analysis of chlorinated pesticides in water 146 Page 73  Advances in GC High-Speed Gas Chromatography: Faster separations in GC can be achieved by using short columns, higher-than- usual carrier gas velocities, and small retention factors. The price to be paid is reduced resolving power caused by increased band broadening and reduced peak capacity. Research workers in the field have been designing instrumentation and chromatographic conditions to optimize separation speed at the lowest cost in terms of resolution and peak capacity. Miniaturized GC Systems For many years, there has been a desire to miniaturize GC systems to the microchip level. Miniature GC systems are useful in space exploration, in portable instruments for field use, and in environmental monitoring. 147 Advances in GC One instrument was specifically designed for measurement of trichloroethylene vapors due to the migration of volatile organic compounds from contaminated soil or groundwater. The miniature GC could be deployed in the field and was capable of sub ppb detection of the vapors. Multidimensional Gas Chromatography In multidimensional GC, two or more capillary columns of differing selectivities are connected in series. Subjecting a sample to separation in one dimension followed by separations in one or more additional dimensions can give rise to extremely high selectivity and resolution. In one implementation, called heart cutting, a portion of the eluent from the first column containing the species of interest is switched to a second column for further separation. This implementation excessively enhances resolution and permit to get rid of specific interferences. 148 Page 74  High-speed chromatogram obtained with isothermal operation (30 C) for 37 s followed by a 35 C/min temperature ramp to 90 C. 149 ‫الح د‬ ‫الذى بنع ه م الص لح ت‬ 150 Page 75