1. M.PRASAD NAIDU Msc Medical Biochemistry, Ph.D Research scholar

2.  2 types  1. GSC2. GLC  GSC is not widely used b/cos limited no of stationary phases available.  Adsorption is the principle  GSC is used only in case where there is less solubility of solutes in stationary phase, which are rare.  GLC only

3.  Partition is the principle  Stationary phase: liquid which is coated on to a solid support  Mobile phase: Gas  Components are separated according to their partition coefficients  Partition coefficient is the ratio of solubility of a substance distributed between two immiscible liquids at a constant temp.

4.  2 important criteria are  1. Volatility: unless a compound is volatile, it cannot be mixed with mobile phase.  2. Thermostability:  All the compounds will not be in the form of vapour.(solids & liquids)  Hence to convert them to a vapour form, they have to be heated.  At that temp the compounds have to be thermostable  If they are not thermostable, the compounds cannot be analysed by GC, since they will be decomposed.

5.  Carrier gas  Flow regulators and flow meters  Injection devices  Columns  Temparature control devices  Detectors  Recorders and integrators

6.  The choice of carrier gas determines the efficiency of chromatographic separation  Eg: H 2, He, N 2, Ar  H 2 : better thermal conductivity, low density  Used in thermal conductivity detector / FID  Demerits:  1. it reacts with unsaturated compounds  2. inflammable  He: excellent thermal conductivity, but expensive  Used in thermal conductivity detector.  N 2 : inexpensive but has reduced sensitivity

7.  Inertness  Suitable to the detector used  High purity  Easily available  Cheap  Less risk of explosion or fire hazards  Should give best performance  Consistent with the required speed of analysis  Compressible, gases are stored under high pressure in cylinders  N2, He are the most commonly used

8.  As carrier gases are stored under high pressure, flow regulators are used to deliver the gas with uniform pressure or flow rate  Flow meters  to measure the flow rate of carrier gas  1. Rotameter:  2. Soad bubble meter:

9.  Gases can be introduced by valve devices  Liquids can be injected through loop or septum devices  Most GC instruments have a high quality rubber septum  Solid samples are dissolved in a suitable solvent and injected through a septum

10.  Glass / stainless steel  Stainless steel columns  long life & can be easily handled without the fear of fragility  But some samples react with them  Hence in such cases, glass columns are used Eg: steroids  Glass columns are inert but highly fragile and are difficult to handle

11.  A) depending on its use:  1. analytical column: 1-1.5m of length & outer diameter of 3-6mm  They are packed columns & are made up of glass or stainless steel  Demerit: Only small quantity of sample can be loaded  2. Preparative columns: larger & large amount of sample can be loaded  3-6m of lengthy, outer diameter 6-9mm

12. Stationary phase NatureTemp Polydimethyl siloxane Non-polar -60 0 -320 0 C Poly(diphenyl) dimethyl siloxane Non-polar bonded phase -60 0 -320 0 C Polycyano propyl phenyl dimethyl siloxane Intermediate polarity Upto 280C Polyalkylene glycol Polar30-220 Polyethylene glycol Polar50-280 PEG modified with Nitroterephthalic acid Polar bonded phase 60-200 Poly bis cyano propyl siloxane Very polar non- bonded phase Upto 250

13.  Long capillary tubing of 30-90m  0.025-0.075cm internal diameter  Stainless steel & coiled  The inner wall is coated with the st. phase liquid (0.5-1µ thin film)  these columns offer least resistance to flow of carrier gas  More efficient than packed columns ( offers more resistance)  Demerit: more sample cannot be loaded

14.  An improved version of Golay or capillary columns  A support material is deposited (1µ) on the inner wall & then coated with a thin film of liquid phase  Have a low resistance to flow of carrier gas  Advantage: more sample load

15.  Preheaters: converts the sample into its vapour form & mix them with mobile phase or carrier gas  Preheaters are present along with injecting devices  Thermostatically controlled oven:  In GC partition is the principle  Since partition coefficient as well as solubility of a solute depends upon temp, temp maintenance in a column is highly essential for efficient separation  Hence column & injecting devices should be maintained at a particular temp.

16.  Isothermal programming: same temp is maintained throughout the process of separation  Linear programming: in which the oven is heated linearly over a period of time  This is required when a sample has a mixture of low bp & high bp compounds  Separation of complex mixtures

17.  Heart of the apparatus  Requirements of an ideal detector:  Applicability to wide range of samples  High sensitivity to even small conc  Rapidity of response  Linearity: i.e., less response to low conc & vice versa  Response should be unaffected by temp, flow rate or characteristics of carrier gas  Non destructive to the sample in case of preparative work  Simple & easy to maintain  inexpensive

18.  Katharometer / Thermal Conductivity Detector (TCD)  Flame Ionization Detector (FID)  Argon Ionization Detector (AID)  Electron Capture Detector (ECD)  Nitrogen Phosphorous Detector (NPD)

19.  Principle: is based upon thermal conductivity difference b/n carrier gas & that of component  TCD has 2 platinum wires of uniform size which form part of Wheatstone bridge  Through one of them, pure carrier gas always flows & through the other the effluents of the column passes  2 Pt wires are heated electrically  When pure carrier gas passes through both or them, there is no diff in temp or resistance & hence baseline is recorded  When a component emerges from the column, it alters the thermal conductivity & resistance of the wire  Hence this produces a diff in resistance  So conductivity b/n wires, which is amplified & recorded as a signal.

20.  The thermal conductivities of some carrier gases:  H 2 =32.7 ; He=33.9 ; N 2 =5.2; CH 4 =6.5; C 6 H 12 =3.0  Advantages:  Applicable to most compounds  Linearity is good  Sample is not destroyed & used in preparative scale  Simple, easy to maintain & inexpensive  Disadvantages:  Low sensitivity  Affected by fluctuations in temp & flow rate  Response is only relative & not absolute  Biological samples cannot be analysed

21.  Based upon the electrical conductivity of carrier gases  At normal temp & pressure, gases act as insulators, but become conductive if ions are present  H 2 is the carrier gas used in FID  If the carrier gas is either N 2 /Ar, it can be mixed with H 2  Anode: Ag gauze placed over the burner tip  Cathode: burner tip made up of Pt capillary  When pure carrier gas alone passes, there is no ionization & no current flows  When a component emerges, no. of ions are produced b/cos of ionization by the thermal energy of the flame  This causes a potential diff & causes a flow of current which is amplified & recorded as a signal

22.  Extremely sensitive & background noise is low  µg quantities can be detected  Stable & insensitive to small changes in the flow rate of carrier gas & water vapour  Responds to most of the org compounds  Linearity is excellent

23.  AID depends on the exitation of Ar atoms to a metastable state, by using radioactive energy.  This is achieved by irradiating the carrier gas with either α- or β- particles  α- particles can be obtained from radium-D  β- particles can be obtained from Sr 90 / H 3  These high E particles ionize the Ar atoms & hence they are exited to metastable state  These molecules collide with the effluent molecules and ionizes them  These ions when reach the detector will cause an increase in current  Thus the components are detected

24.  Advantages:  Responds to most of the org compounds  Sensitivity is very high  Disadvantages:  Response is not absolute & it is relative  Linearity is poor  Sensitivity is affected by water & is much reduced for halogenated compounds  The response varies with the temp of the detector  High temp like 240 0 C, voltages of 1000V or less are usually necessary

25.  ECD has 2 electrodes  Column effluent passes b/n them  One of the electrode is treated with a radio active isotope which emits electrons as it decays.  These emitted electrons produce 2 o electrons which are collected by the anode, when a PD of 20V is applied b/n them  When carrier gas alone flows through, all the 2 o electrons are collected by the +vely polarised electrode  Hence a steady baseline is recorded  Effluent molecules which have affinity for electrons, capture these e- when they pass through the electrodes  Hence the amount of steady state current is reduced  This diff is amplified & recorded as output signal

26.  The carrier gas used in this detector depends upon the e- affinity of the compounds analysed  For compounds with high e- affinity, Ar is used  For low e- affinity, N 2, H 2, He or CO 2 can be used  Advantages: highly sensitive (10 -9 )  Disadvantage: ECD can be used only for compounds with e- affinity  Halogenated compounds, pesticides etc can be detected by ECD

27. Detector Katharo meter/ TCD FIDAIDECD Min. detectable conc %v/v 10 -6 10 -11 10 -12

28.  Recorders : to record the responses  They record the baseline & all peaks obtained with respect to time  Retension time for all the peaks can be found out from such recordings, but the area of individual peaks cannot be known  Integrators: improved version of recorders with some data processing capabilities  Can record the individual peaks with Rt, height & width of peaks, peak area, % of area, etc  Int provide more information on peaks than recorders