Advance instrumental analysis

1. Properties that determines the equilibrium partition The property that determines the equilibrium partition between the mobile and stationary phase is the polarity of the solute particle and polarity of the stationary phase. Mobile phase here is just to carry the vaporized solute molecules. Molecules having grater attraction towards stationary phase will retain in the column for the long time. Viscosity of the mobile phase effect separation and resolution but do not determine the equilibrium partition. 2. Comparing nitrogen, hydrogen and helium.

There are some characteristics of these curves Height eq plate is inversely proportional to number of theoretical plates and number of theoretical plates is proportional to efficiency. From graph lowest value is achieved by more or less each gas but the slope of curve N2 is Steeper than the slope of He and H2 which means the variation in velocity of mobile phase will cause grater change in efficiency during the process and it will effect reproducibility and separation. So best gas is the gas have low slope which is hydrogen but its dangerous so we use helium gas.

3. Injector recommended for low concentration samples The choice of injector actually depends on a. The characteristics of specimen or residues (gas, liquid or solid) b. The quantity and characteristics of analytes to be separated (high or low) c. The temperature and nature of stationary phase (column characteristics) There are four types of injection system a. Magabore (packed column) injection system (for large volume and high concentration samples) b. Split mode injection system (high concentration) c. Split less mode injection system (low concentration) d. Cold on-column injection system ( thermally labile compounds and having high boiling point difference) Usually splitless mode of injection is used for trace analysis because the sample is first vaporized with less flow then a high flow of gas is purged to send all the solute in to the column. This injection system has its own band broadening problems that can be solved by solvent effect of cold trapping. 4. Head space extraction This technique is used for extracting volatile compounds which can be directly injected to the system. The volatile compounds present in a closed container are in equilibrium with liquid and gas phase. An air tight syringe can be used to take the sample from the head space of the container and it can be injected to the column using a loop injection valve system. In first step the sample is injected in the loop and the position is changed so the carrier gas can flow through the loop and carries all samples to column. This is the most efficient inlet system. 5. Solid phase micro extraction technique In this technique a syringe with fused silica fiber with a liquid phase coating is immersed to sample containing analytes, analytes will adsorbed or solubilize in the solid or liquid coating on the fused silica. In this method the extraction efficiency is very less.

6. Comparing packed and capillary column Packed columns are filled with stationary phase while in capillary columns the stationary phase is coated on the wall of the column. There are many factors that affect the efficiency of column in which one important factor is eddy diffusion. In packed column there is more possibility of eddy diffusion then in capillary column because different molecules will cover different distance while passing through the column which decreses it efficiency. 7. Resolution, capacity and applicability of capillary column There are commonly three types of capillary column used a. Thin film narrow bore b. Tick film narrow bore c. Thick film wide bore Thinner the coating more efficient is the column and it will also increase the resolution of the column but the capacity of the column will be less and also the columns will not be sensitive. So we can say that thin film narrow bore columns have more resolution but less capacity and things will be reverse for Thick film wide bore. It also depends on some factors discussed in the equation below K = k x r /2d where k is retention factor, K is eq constant, r is radius of the column, and

d is thickness of the stationary phase. K remains constant at contant temperature so increasing d and decreasing r will increase k 8. Stationary phase used for GC columns A lot of stationary phase can be used keeping in mind that the stationary phase should have low vapor pressure, thermally stable, low viscosity, and it should be chemically inert. Usually polysiloxane is used for non-polar compounds and polyethylene glycol is used for polar compounds but is very less stable. The R group in polysiloxane may be a methyl, phenyl, or a mixture of methyl and phenyl depending on the solute you want to separate.

9. Selectivity in stationary phase of GC Methyl PSX is commonly selective for long chain non polar hydrocarbon. Bc it can only produce dispersion forces. Phenyl PSX is used for aromatic compounds just bc of its pi pi interactions. It also has strong dispersion forces and very little hydrogen bonding. Cyano propyle PSX is used for insaturated fatty acids, alcohols and free acids. PEG has strong polar interaction so it can be used essential oil free acids and glycols. 10. Columns bleed The phenomena in which the stationary phase starts eluting from the column is known as column bleed. It may be due to many reasons like increase in temperature from the limit so that stationary phase thermally decomposed and elute from the column. It is also due to some contamination in mobile phase which dissolve the stationary phase. The base line is not remain at zero and it will give you some humps (not peaks). 11. Selection for detectors a. Polycyclic aromatic hydrocarbons: these compounds can be analyzed by FID detector as it is best for hydrocarbon but it is moderate sensitive and sensitivity depends on no of carbons and flow rate its sensitivity is in pg and range in 107. b. Halogenated compounds: these compounds can be analyzed using electron capture detector as these compounds are electron rich electronegative compounds only sensitive to electron capturing groups like CL Br etc and range is 103. c. Amines: nitrogen phosphorus detector is very selective for the compounds containing nitrogen and phosphorus so ammines can be analyzed by it.500 times better sensitive then FID but not for organic compounds 12. Two dimensional GC Two dimensional GC is a technique in which two columns of different lengths are used for separating compounds at different stages. In the first column the compounds are separated according to their boiling points. If the compounds have boiling point difference greater than 30 degree then they can be easily separated. When the components elute from the first column they goes to thermo electric cooler where they are condense to concentrate and then heats up to send the components to other column

where they are separated by their solubility in stationary phase. Final chromatogram that we obtain is a complex chromatogram and software is used to resolve it. 13. Types of chromatogram in GC x GC In this chromatogram we obtain a complex line. Bc each component separated in first column is again separated in second column. The three dimensional view and upper view can explain the retention of components. 14. Peak area vs peak height Peak height has many problems like height is changed after every degree change in temperature. Also if we change gas flow the height will change. Whenever there is peak broadening, peak height is affected by peak broadening. 15. Internal and external standard In external standard we make standard solution of different concentrations which contains only our own analytes and graph is made bw con and signal. While in internal sgtandard we add another compound similar to our analytes in same amount in each standard and smaple. And we plot a graph bw signal of standard to the signal of In standard and concentration of standards. The reason for using internal standard is the sample should face the same conditions as our standard are facing. It is used when reproducibility is required.