GC_PPT

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gas chromatography

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GAS CHROMATOGRAPHY:

GAS CHROMATOGRAPHY By :- R.D ivya Reddy, Roll no.:-02, M.Pharm . Kvk college of pharmacy.

What is Gas Chromatography?:

What is Gas Chromatography? It is one of a chromatographic technique in which the components of a mixture are separated by using gas as mobile phase and solid /liquid as stationary phase.

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Introduction Principle Criteria of compounds for analysis Instrumentation Derivatisation of sample Parameters Applications OVERVIEW

INTRODUCTION:

Simple & efficient separation technique GC is of two types:- GSC (gas solid chromatography) GLC (gas liquid chromatography Gas → Mobile Phase(M.P) Solid / Liquid → Stationary Phase(S.P) GSC not used because of limited no. of S.P GSC principle is ADSORPTION GLC principle is PARTITION INTRODUCTION

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Sample to be separated is converted into vapour And mixed with gaseous M.P Component more soluble in the S.P → travels slower Component less soluble in the S.P → travels faster Components are separated according to their Partition Co-efficient

DEVELOPMENT OF GC BY?:

DEVELOPMENT OF GC BY ? The Father of modern gas chromatography & The Nobel Prize winner John Porter Martin(1952) , who also developed the first liquid-gas chromatograph. (1951)

PRINCIPLE:

GSC:- The principle in GSC is adsorption , the components are separated according to their relative affinities towards their stationary phase. Based on (1/n) x/m=kc --->freaundlich law. x/m=k 1 c+k 2 c---->langmuir law. GLC:- The principle in GLC is partition , the components are separated according to their partition co-efficient values. x/m=kc---->henrys law of partition. PRINCIPLE

CRITERIA OF COMPOUNDS FOR ANALYSIS:

The components must posess :- Volatility :- unless a compound is volitile it cant be mixed with mobile phase. Thermostability :- the solids/liquids------->gases they are heated to greater temp were the compounds must be stable. they must not decompose. CRITERIA OF COMPOUNDS FOR ANALYSIS

Instrumentation:

Instrumentation

INSTRUMENTATION:

Carrier gas. Flow regulators & flow meters. Injection devices. Columns. Temperature control devices. Detectors. Recorders & integrators. INSTRUMENTATION

a)CARRIER GAS:-:

The carrier gases generally used in GC:- Hydrogen better thermal conductivity disadvantage: it reacts with unsaturated compounds & inflammable Helium excellent thermal conductivity it is expensive Nitrogen reduced sensitivity it is inexpensive a)CARRIER GAS:-

Requirements of a carrier gas:

Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance Requirements of a carrier gas

b)Flow regulators & Flow meters:

Deliver the gas with uniform pressure/flow rate F low meters:- Rota meter & Soap bubble flow meter a) Rota meter Placed before column in-let, it has a glass tube with a float held on to a spring. The level of the float is determined by the flow rate of carrier gas . b)Flow regulators & Flow meters

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Rotameter:-

b)Soap Bubble Meter:

◊ Similar to Rota meter & instead of a spring, soap bubble formed indicates the flow rate. b)Soap Bubble Meter

c)Injection Devices:

Gases can be introduced into the column by valve devices liquids can be injected through loop or septum devices c)Injection Devices

d)COLUMNS:

Important part of GC Made up of glass or stainless steel. Glass column- inert , highly fragile.(steroids) Steel is mostly used . COLUMNS can be classified Depending on its use 1. Analytical column 1-1.5 meters length & 3-6 mm in diameter 2. Preparative column 3-6 meters length, 6-9mm in diameter d)COLUMNS

Depending on its nature:

1.Packed column: columns are available in a packed manner S.P for GLC: polyethylene glycol, esters, amides, hydrocarbons, polysiloxanes… 2.Open tubular or Capillary column or Golay column Long capillary tubing 30-90 M in length Uniform & narrow d.m of 0.025 - 0.075 cm Made up of stainless steel, copper, nylon & glass Disadvantage: more sample cannot loaded Depending on its nature

3.SCOT columns (Support coated open tubular column:

Improved version of Golay / Capillary columns, have small sample capacity Made by depositing a micron size porous layer of supporting material on the inner wall of the capillary column Then coated with a thin film of liquid phase 3.SCOT columns (Support coated open tubular column

Columns:

Columns Packed Golay

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Thermostaticaly Controlled oven

e)Temperature Control Devices:

Preheaters : convert sample into its vapour form, present along with injecting devices. Thermostatically controlled oven : temperature maintenance in a column is highly essential for efficient separation. Two types of operations Isothermal programming:- maintained same temp through out the process. Linear programming:- the temp is increased continously or in step wise manner, more efficient for separation. e) Temperature Control Devices

Detector Systems:

Detectors are the devices which convert physical properties of compound to electrical signals. The generally used are:- Thermal conductivity detectors. Flame ionisation detectors. Argon ionisation detectors. Electron capture detectors. Detector Systems

Thermal Conductivity Basics:

Thermal Conductivity Basics When the carrier gas is contaminated by sample , the cooling effect of the gas changes. The difference in cooling is used to generate the detector signal. The TCD is a nondestructive, concentration sensing detector. A heated filament is cooled by the flow of carrier gas. Flow Flow

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When a separated compound elutes from the column , the thermal conductivity of the mixture of carrier gas and compound gas is lowered. The filament in the sample column becomes hotter than the control column. The imbalance between control and sample filament temperature is measured by a simple gadget and a signal is recorded Thermal Conductivity Detector

Flame Ionization Detector:

Destructive detector The effluent from the column is mixed with H & air, and ignited. Organic compounds burning in the flame produce ions and electrons , which can conduct electricity through the flame. The ions collected on collector or electrode and were recorded on recorder due to electric current. Flame Ionization Detector

Argon ionization detector:

Depends on the excitation of argon atoms to a metastable state, by using radioactive energy. Argon → irradiation Argon + e - → collision Metastable Argon → collision of sub. → Ionization → ↑ Current ADVANTAGES 1.Responds to organic compounds 2.High sensitivity DISADVANTAGES 1.Response is not absolute 2.Linearity is poor 3. Sensitivity is affected by water Argon ionization detector

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ELECTRON CAPTURE DETECTOR The detector consists of a cavity that contains two electrodes and a radiation source that emits  - radiation (e.g. 63 Ni, 3 H) . If a compound is present that contains electronegative atoms, those electrons are captured and negative ions are formed, and rate of electron collection decreases

RECORDERS & INTEGRATORS:

RECORDERS :- Record the baseline and all the peaks obtained. INTEGRATORS:- Record the individual peaks with Rt, height…. RECORDERS & INTEGRATORS

Derivatisation of sample:

Treat sample to improve the process of separation by column or detection by detector. They are 2 types Precolumn derivatisation Components are converted to volatile & thermo stable derivative. Conditions - Pre column derivatisation Component ↓ volatile Compounds are thermo labile ↓ tailing & improve separation Derivatisation of sample

Post column derivatisation:

Improve response shown by detector Components ionization / affinity towards electrons is increased Pretreatment of solid support To overcome tailing Generally doing separation of non polar components like esters, ethers… Techniques : 1. use more polar liquid S.P 2. Increasing amt. of liquid phase 3.Pretreatment of solid support to remove active sites. Post column derivatisation

Parameters used in GC:

Retention time (Rt) It is the difference in time b/w the point of injection & appearance of peak maxima. Rt measured in minutes or seconds (or) It is the time required for 50% of a component to be eluted from a column Retention volume ( Vr ) It is the volume of carrier gas which is required to elute 50% of the component from the column. Retention volume = Retention time ˣ Flow rate Parameters used in GC

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Separation factor (S) Ratio of partition co-efficient of the two components to be separated. If more difference in partition co-efficient b/w two compounds, the peaks are far apart & S Is more. If partition co-efficient of two compounds are similar, then peaks are closer Resolution (R) The true separation of 2 consecutive peaks on a chromatogram is measured by resolution It is the measure of both column & solvent efficiencies R= 2 (Rt 1- Rt 2) W 1 +W 2

Resolution:

Resolution

Theoritical plate:

Theoritical plate is an imaginary unit of a column where solute distribution between SP and MP reaches equilibrium. HETP:- Height equivalent to theoritical plate. if HETP is less -------------- >column efficiency is more If HETP is more------------->column efficiency is less Theoritical plate

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HETP= L (length of the column) N (no of theoretical plts) HETP is given by Van Deemter equation HETP = B + u A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column B = Molecular diffusion, depends on flow rate C = Effect of mass transfer,depends on flow rate u = Flow rate

Efficiency ( No. of Theoretical plates):

It can be determined by using the formula n = 16 Rt 2 w 2 N = no. of theoretical plates Rt = retention time W = peak width at base The no. of theoretical plates is high, the column is highly efficient For G.C the value of 600/ meter Efficiency ( No. of Theoretical plates)

Asymmetry Factor:

Chromatographic peak should be symmetrical about its centre If peak is not symmetrical- shows Fronting or Tailing FRONTING Due to saturation of S.P & can be avoided by using less quantity of sample TAILING Due to more active adsorption sites & can be eliminated by support pretreatment, Asymmetry Factor

Fronting and Tailing:

Fronting and Tailing

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Asymmetry factor (0.95-1.05) can be calculated by using the formula AF=b/a b & a calculated at 5% or 10% of the peak height

Applications of G.C:

G.C is capable of separating, detecting & partially characterizing the organic compounds particularly when present in small quantities. 1, Qualitative analysis RT & RV are used for the identification. Checking the purity of a compound Compare the chromatogram of the std . & that of the sample . 2) Quantitative analysis:- peak areas and peak vol. are to be measured. 3)The detection of steroid drugs used by atheletics in competitions. 4)In analysis of foods,identification of lipids, fats, carbohydrates, preservatives, flavors……………….. 4)Dairy products can be analysed for aldehydes and ketones(rancidity). Applications of G.C

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5), used for analysis of drugs & their metabolites. 6)Separation and identification of polycyclic aromatic compounds,chlorinated pesticides(DDT),organophosporous & sulpur compounds. 7)Contaminants in environment.

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Tentative Identification of Unknown Compounds Response GC Retention Time on Carbowax-20 (min) Mixture of known compounds Hexane Octane Decane 1.6 min = RT Response Unknown compound may be Hexane 1.6 min = RT Retention Time on Carbowax-20 (min)

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