Chromatography Techniques PDF

## Chromatographic Techniques ### Chromatographic Techniques can be used for? * **A. Separation** * **B. Identification & determination of chemical components in complex mixtures** * **C. Both of the above** * **D. Only Option A is correct** **Answer:** C. Both of the above **Explanation:** Ch...

## Chromatographic Techniques ### Chromatographic Techniques can be used for? * **A. Separation** * **B. Identification & determination of chemical components in complex mixtures** * **C. Both of the above** * **D. Only Option A is correct** **Answer:** C. Both of the above **Explanation:** Chromatographic techniques are used for separating a mixture and identifying the individual components of that mixture. They are based on differences in the partitioning of components between a stationary phase (solid or liquid) and a mobile phase (liquid or gas). **Chroma-Colour** **Graphein- To write** **Mikhail Tswett** - Russian botanist used this technique to separate out various plant pigments & separation was observed as colour bands. **Chromatographic techniques are now used for** * **Separation** * **Identification** * **Determination of chemical components in complex mixtures** **Diagram:** The diagram shows a chromatogram of plant pigments with the following identified components: * Carotene * Phaeophytin * Chlorophyll A * Chlorophyll B * Lutein * Vi-axanthin * Neoxanthin ### In chromatography, Mobile Phase are generally * **A. Solid/Liquid** * **B. Liquid/Gas** * **C. Solid, Liquid & Gas** * **D. All of the above options are correct** **Answer:** D. All of the above options are correct. **Explanation:** In chromatography, the mobile phase can be solid, liquid, or gas. The selection of the mobile phase depends on the nature of the sample and the type of chromatography being performed. **2 Phases** * **Stationary/ Fixed Phase:** For sorption & desorption. May be solid/liquid * **Mobile Phase:** Liquid/gas ### For volatile materials, Which chromatographic technique is the first choice? * **A. Affinity chromatography** * **B. HPLC** * **C. TLC** * **D. Gas chromatography** **Answer:** D. Gas chromatography **Explanation:** Gas chromatography is the technique of choice for volatile materials because it can separate and identify different components based on their boiling points. **Classification of Chromatographic Techniques** **Mainly 5 types** * **Adsorption Chromatography** * **Partition Chromatography** * **Ion Exchange** * **Size exclusion/pore penetration** * **Affinity Chromatography** **Note: How to select a particular mode of chromatography depends upon complexity of sample, physical and chemical properties of compounds to be separated.** **Example:** * **Volatile materials & stable in gas phase:** Gas Chromatography (GC) is the choice. * **High molecular weight compounds:** Proteins, triglycerides, polymers-Size exclusion is the best choice. * **Quantitative analysis:** HPLC * **Ionic compounds:** Amino acids-Ion exchange mode of chromatography is better option. ### Gas Chromatography #### What is Gas Chromatography? (GC) Gas chromatography (GC) is an analytical technique applicable to gas, liquid, and solid samples (components that are vaporized by heat). **Chemical derivatization of the target analytes that are usually polar, thermally labile, or hydrophilic, is necessary**. **Detection of polar, non-volatile metabolites requires chemical derivatization prior to analyses**. To be amenable to analysis by GC, compounds must have a relatively high vapor pressure. #### Types of Gas Chromatography (GC) The stationary phase may be a solid or a liquid. Depending on whether the stationary phase is a solid or a liquid at its operating temperature, GC is classified as gas-solid chromatography (GSC) or gas-liquid chromatography (GLC). **Gas-liquid chromatography is the method most commonly used to separate organic compounds.** #### Compounds Unsuitable/Cannot be analyzed with Gas Chromatography (GC) ##### Compounds That Cannot Be Analyzed * Compounds that do not vaporize (inorganic metals, ions, and salts) * Highly reactive compounds and chemically unstable compounds (hydrofluoric acid and other strong acids, ozone, NOx and other highly reactive compounds) ##### Compounds That Are Difficult to Analyze * Highly adsorptive compounds (compounds containing a carboxyl group, hydroxyl group, amino group, or sulphur) * Compounds for which standard samples are difficult to obtain (Qualitative and quantitative analyses are difficult.) #### Overview of Gas Chromatography * When a mixed solution sample is injected into the GC system, the compounds contained in the sample, including the solvent components, are heated and vaporized within the sample injection unit. * With GC system, the mobile phase, referred to as the carrier gas, always flows in sequence from the sample injection unit to the column, and then to the detector. The target components that were vaporized in the sample injection unit are transported by the carrier gas to the column. * Once in the column, the mixture of compounds is separated into the various components, and the amount of each compound is then measured by the detector. * The detector converts the amount of each compound into an electrical signal, and sends these signals to a data processing unit. The data obtained enables determination of the compounds contained in the sample, and in what amounts. #### Diagram The diagram shows a basic schematic of a gas chromatograph with the following components: * **Liquid Sample:** This is the sample that is being analyzed. * **Sample Injection Unit:** This is where the liquid sample is injected into the system. * **Carrier Gas:** This is an inert gas that is used to carry the sample through the column. * **Column:** This is where the separation of the components takes place. * **Detector:** This is where the separated components are detected. * **Data Processing Unit:** This is where the data from the detector is processed and displayed. #### Gas Chromatography: Instrumentation There are three main GC system components: * **Sample Injection Unit:** This heats the liquid sample and vaporizes it. * **Column:** This is used to separate each compound. * **Detector:** This detects the compounds and outputs their concentrations as electrical signals. #### Diagram The image shows a more detailed schematic of a GC system, which includes: * **Cylinder:** This contains the carrier gas. * **Carrier Gas Flow Controller:** This controls the flow rate of the carrier gas. * **Sample Injection Unit:** This injects the sample into the system. * **Column Oven:** This heats the column to control the separation process. * **Column:** This is where the separation takes place. * **Detector:** This detects the separated components. * **Data Processing Unit:** This processes and displays the data from the detector. #### Carrier Gas/Mobile Phase * **He - Most commonly used** * Inert gas (N₂, He, Ar, H₂, etc.) as the mobile phase. * The most commonly used gas in GC is helium because it is inert, nontoxic, non-flammable, and provides high optimum linear velocity. * **Nitrogen** * Nitrogen is also a common carrier gas in GC; the main drawback of this gas is its lower optimal linear velocity compared to helium or hydrogen, leading to longer analysis times. This makes nitrogen the least desirable carrier gas in GC. * **Carrier Gas** * This is needed for the transfer of the injected sample to the separation column. They are also responsible for the subsequent transfer of separated components to the detector. Common examples: Nitrogen, helium, or hydrogen. * **Fuel Gas** * They support the flame in Flame ionization detector (FID) detector such as Hydrogen. * **Zero Air** * These are the purified air that plays the role of oxidant to support the combustion of flame in the detector. Before being led to the gas chromatographic system, the above three are intermixed in the desired proportion. #### Table 7: Typical gas chromatography detectors and their detection limits. | Type of Detector | Applicable Samples | Detection Limit | |-------------------------|-----------------------------------------------|-------------------| | Mass Spectrometer (MS) | Tunable for any sample | .25 to 100 pg | | Flame Ionization (FID) | Hydrocarbons | 1 pg/s | | Thermal Conductivity (TCD) | Universal | 500 pg/ml | | Electron-Capture (ECD) | Halogenated hydrocarbons | 5 fg/s | | Atomic Emission (AED) | Element-selective | 1 pg | | Chemiluminescence (CS) | Oxidizing reagent, Vapor and gaseous compounds | Dark current of PMT | | Photoionization (PID) | Vapor and gaseous compounds | .002 to .02 µg/L | #### Applications of GC * Content Estimation in biodiesel * Toxicology-Pesticides & Herbicides * Metabolomics * Forensic science to separate substances of analytical interest. * GC is the primary technique for the analysis of fire residues. Petroleum products. * The use of GC in the detection and quantitation of alcohol in drunk-driving cases. * Essential role in the analysis of persistent organic pollutants, such as dioxins, polychlorinated biphenyls (PCBs), brominated flame retardants, polychlorinated naphthalenes, perfluorosulfonates, amides, perfluorotelomers, neutral perfluorinated compounds, short-chain chlorinated paraffins, environmental endocrine disruptors, sunscreens, and synthetic musk. ### Which chromatographic technique is based on the principle of Adsorption * **A. Column chromatography** * **B. Thin layer chromatography** * **C. Gas solid chromatography** * **D. All of the above** **Answer:** D. All of the above **Explanation:** Adsorption chromatography is a type of chromatography that uses a solid stationary phase to adsorb the components of a mixture. The stationary phase is usually a silica gel or alumina. The mobile phase is a liquid or gas. All of the above techniques are based on the principle of adsorption. #### Adsorption chromatography * Stationary phase - Solid * Mobile Phase - Liquid/Gas **Examples:** * **Column Chromatography** * **Thin layer chromatography-TLC** * **Gas solid chromatography** * **Liquid column chromatography** ### Most of the time reverse-phase chromatography is preferred as it has…...Stationary phase * **A. Non-polar stationary phase** * **B. Polar stationary phase** * **C. Polar mobile phase** * **D. Options A & C both are correct** **Answer:** D. Options A & C both are correct. **Explanation:** Reverse-phase chromatography is a type of chromatography that uses a non-polar stationary phase and a polar mobile phase. This is the most commonly used type of chromatography because it can separate a wide variety of compounds. The non-polar stationary phase is usually a C18 or C8 bonded silica gel. The polar mobile phase is usually a mixture of water and an organic solvent like methanol or acetonitrile. #### Normal Phase Vs Reversed phase chromatography **Normal Phase** * Stationary phase - Polar(Water or methanol) * Mobile Phase - Nonpolar(Hexane) *Retention of polar compounds & elution of nonpolar compounds* **Reversed Phase** * Stationary phase - Non polar * Mobile Phase - Polar *Retention of Nonpolar solutes & elution of polar solutes* ### Choose the correct statement about ion exchange chromatography * **A. Ion exchange resins are used as stationary phase** * **B. Always liquid is preferred as mobile phase** * **C. Method of choice for analysis of inorganic ions** * **D. All of the above are correct** **Answer:** D. All of the above are correct **Explanation:** Ion exchange chromatography is a type of chromatography that uses a stationary phase that has charged groups. This charged stationary phase can be used to separate ions based on their charge and polarity. ### With reference to the HPLC chromatography, chromatogram refers to * **A. Instrument** * **B. Peak graph** * **C. Both of the above** * **D. None of the above are correct** **Answer:** B. Peak graph **Explanation:** Chromatogram is the visual representation of the separation of a mixture obtained by a chromatographic technique. In HPLC (High-Performance Liquid Chromatography) chromatogram is obtained on the basis of intensity of the signal of analyte coming out of the column and plotted against time. So, it is a peak graph representing the elution of different analytes. #### Chromatograph Vs Chromatogram * Chromatograph - Instrument * Chromatogram - Peak Graph ### High performance liquid chromatography: HPLC - High pressure upto 5000 psi required to force the mobile phase & perform this chromatography. - Purity of compound matters - Analytical grade. - Mobile phase filtered to remove particulate matters. - Degassing & sonication also performed. - Mechanical Pumps - Reciprocating piston type - Delivers at constant flow rate. - Pneumatic Pumps - Produce constant pressure, pulseless operation. ### Components/Instrumentation of HPLC * **Sample** * **Reservoir/Mobile Phase** * **Pump** * **Precolumn** * **Column:** Heart of HPLC Separation. C4, C8 & C18 are nonpolar phases. * **Detector** * **Monitor** ### HPLC Method development * Flow rate - ml/minute * Detector - * Mobile phase - * Stationary Phase - * HPLC Column - Silica Gel. * **Role of Precolumn/Guard column:** To avoid shrinkage of silica material in column & prevention of contamination by traping particulate matters. * Silica generally gets dissolved in solvents having pH below 2 & above pH 7 - creates shrinkage, void spaces and loss of resolution. * Does not affect separation process because of short length (2-10 cm) ### HPLC Method development & Validation * **Microliter syringe** ### HPLC Column * **Heart & act as stationary phase.** * **Glass columns:** Chloride containing buffers as they can deteriorate steel columns. * **Stainless steel columns:** All organic & aqueous buffers. * **Internal diameter:** 2-4.6 mm. * **Particle size of HPLC column:** 5 Micrometre Good for separation due to column efficacy & back pressure. * **Bonded phase of column:** C1, C4, C8, C18. * Silica based adsorbents modified with trimethylchlorosilane (C1) & Butyldimethylchlorosilane (C4). * Octyl(C8) & Octadecyl(C18). ### Detectors for HPLC * **1.Optical Detectors** * UV/Visible_Photodiode array/PDA detectors * Fluorescence detectors * **Universal detectors:** Give response to all samples. Detects low level of compounds. * **2.Mass Spectroscopy detectors** * Time of Flight/MALDI detectors * Mass spectrometry ### In HPLC chromatography, Method development involves * **A. Selection of Mobile Phase** * **B. Selection of column** * **C. Optimization of flow rate** * **D. All of the above are correct** **Answer:** D. All of the above are correct. **Explanation:** * **Selection of Mobile Phase:** mobile phase chemistry significantly influences the separation. * **Selection of column:** The column is crucial for maintaining a high resolution of separation, and the specific properties of the column should be chosen based on the sample to be analyzed. * **Optimization of flow rate:** The flow rate is an important parameter that influences the speed of the separation and the resolution of the peaks. ### Which is being included in HPLC Instrumentation? * **A. Stationary + Mobile phase** * **B. Pump, Precolumn & column** * **C Detector & Monitor** * **D. All of the above** * **E. None of the above** **Answer:** D. All of the above **Explanation:** * **Stationary + Mobile phase:** This forms the basis of the separation process in HPLC. * **Pump, Precolumn & column:** These are essential for the delivery and separation of analytes in the system. * **Detector & Monitor:** These components are responsible for detecting the separated analytes and displaying the data. ### Isocratic elution simply means * **A. Composition of mobile phase remains the same during run of a sample** * **B. Composition of the mobile phase changes during run of a sample** * **C Use of One mobile Phase** * **D. All of the above** **Answer:** A. Composition of mobile phase remains the same during run of a sample. **Explanation:** Isocratic elution is a technique in chromatography where the composition of the mobile phase remains constant throughout the entire separation process. It is used to elute different components of the sample at a constant rate. ### Choose the right one from below mentioned options * **A. TLC is used to isolate non volatile mixtures** * **B. Silica gel is the preferred stationary Phase** * **C TLC is limited to qualitative analysis, and it can not be used for quantitative analysis.** * **D. All of the above** **Answer:** D. All of the above **Explanation:** All the statements are correct. TLC is used for separating non-volatile mixtures. It is a simple and versatile technique used for qualitative analysis. Silica gel is a commonly used stationary phase due to its unique properties, including its adsorption capabilities and surface area. TLC can identify compounds but is not usually used for quantitative analysis. #### Thin Layer Chromatography: TLC * Used to isolate non-volatile mixtures. * The experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent material. The material usually used is aluminium oxide, cellulose, or silica gel. * On completion of the separation, each component appears as spots separated vertically. * The factors affecting retardation factor are the solvent system, amount of material spotted, adsorbent and temperature. * **Diagram:** The diagram shows a TLC plate with a green compound that has been separated into individual spots using a solvent system. #### Each spot has a retention factor (Rf) expressed as: * **Rf = dist. travelled by sample / dist. travelled by solvent** #### Diagram: The diagram shows a TLC plate with different spots representing different compounds separated vertically on the plate. A red spot is marked as the sample. The distance travelled by the sample spot is measured from the origin to the center of the spot. The distance travelled by the solvent front is measured from the origin to the solvent. #### Application of TLC * **TLC is limited to qualitative analysis, and it cannot be used for quantitative analysis.** * **Product identification** * **Product Purity** * **Medicines like local anaesthetics, analgesics, sedatives, hypnotics, anticonvulsant tranquilizers, and steroids go through the TLC procedure for their qualitative testing.** * **TLC also finds its use in Biochemical analysis. Here, it can be used for biochemical metabolites' separation from urine, blood plasma, serum, and body fluids.** * **The cosmetic industry also uses TLC for checking the presence of preservatives in products.** #### Table 7: Some frequently used reagents and their characteristics in TLC | Reagent | Works well for | Colors | Notes | |-------------------------------------------|----------------------------------------|-------------------------------------------|---------------------------------------------------------------------------------------------| | Iodine | Unsaturated and aromatic compounds | Brown spots | Not permanent | | Sulfuric acid | General stain | Brown or black spots | | | Chromic acid | For difficult to stain compounds | Black spots | | | UV light | Compounds with extended conjugation | Pink on light green background | Only visible under UV light | | Cerium sulfate | Good general stain, very well for alkaloids | Purple | | | Ferric chloride | Phenols | Yellow/orange | | | Ninhydrin | Amino acids, amines | Purple | also called "DNP" | | 2,4-Dinitrophenylhydrazine | Aldehydes, ketones | Yellow on purple | | | Vanillin | Good general stain, very well for hydroxyl or carbonyl compounds | Colors vary | | | Potassium permanganate | Works well for all compounds that can be oxidized | Yellow or light brown on purple background | at rt. for alkenes and alkynes upon heating for alcohols, amines, sulfides | | Bromocresol Green | Carboxylic acids (pK<5) | Yellow spot on blue background | | | Cerium molybdate (CAM, 'Hanessian's Stain', Ceric staining) | Good general stain, very well with polyhydroxylated and carbonyl compounds | Blue or green spot | Upon heating, very sensitive! | | p-Anisaldehyde | Good general stain, particularly sensitive towards nucleophiles | Varying colors on light pink plate upon heating | Does not work with alkenes, alkynes or aromatic system unless functional groups are present. | | Phosphomolybdic acid (PMA) | Very sensitive | Dark green spot on light green plate | Sensitivity can be enhanced by use of cobalt(II) choride. | | Ehrlich’s Reagent (Dimethylaminobenzaldehyde) | Indoles, amines | Pink or red-violet | | | Dragendorff-Munier Stain | Amines even the ones that are low in reactivity | Various colors | |