3. A SEMINAR ON Chromatography Introduction and Instrumentation GUIDED BY: BY: PATEL AKSHAY J.D.Patel M.PHARM- 1(ph’ceutics) (head of department) ROLL NO-05 Department of Pharmaceutics NOOTAN PHARMACY COLLEGE,VISNAGAR
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8. It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better). If the length of the column is L , then the HETP is HETP = L / N The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution; where w 1/2 is the peak width at half-height. As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture.
9. A- Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths. B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion. C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.
35. Refractive Index Detector : Two types : (a) Reflection type (b) Deflection type. A beam of light is reflected at eluent prism interface. Other is refracted and that beam after passing through collimating lens is falling on photocell, which measures its intensity. When solute enters the eluent, the RI of eluent changes. As a result the intensity of beam reflected at eluent prism interface changes. This leads to change in intensity of beam refracted which is measured by photocell. Thus change in intensity concentration of solute of refracted beam
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38. Stationary phases : They can be either liquid or solid. If liquid is used it is coated on inert solid, but there are problems in it. Hence, bonded phase supports prepared.
39. Silica is heated in dilute acid for a day or two to generate silonal group. As follows: This is then treated with an organochlorosilane. R = long alkyl chain of 8 or 18 carbon then Nonpolar (Reversed phase) R = -(CH 2 )n-CN, then polar (Normal phase) = -(CH 2 )n-NH 2 Stable upto pH 2 & 9 and upto 80 0 C. How they retain solute molecules is not certain.
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41. Normal phase and Reversed phase Chromatography : Initially the stationary phase used to be polar and mobile phase used to be non-polar. This combination became popular as normal phase chromatography. Later the use of non-polar stationary phase and polar stationary phase started. This was reverse to the established combination and hence it is called reversed phase chromatography.
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44. Solvents Polarity Index B. P. Cyclohexane 0.04 81 n-hexane 0.1 69 Toluene 2.4 110 THF 4.0 66 Ethanol 4.3 78 Ethyl acetate 4.4 77 Methanol 5.1 65 Acetonitrile 5.8 82 Nitromethane 6.0 101 Water 10.2 100
45. Isocratic & Gradient elution : If comp. of mobile phase does not change during elution, it is isocratic. If comp. changes then Gradient. Need for Gradient elution : If we want to separate mix. of 10 solutes, 5 N.P. and 5 highly polar and NP mobile phase used then NP solutes eluted in reasonable time but polar solutes take long time to come out. If mobile phase is polar then NP solutes eluted so quickly that no resolution but now polar solutes come out in reasonable time. If we make mobile phase gradually from NP to polar then the problem is solved and that is called gradient elution.
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47. Varian HPLC System 9010 Solvent Delivery System 9050 Variable UV/Vis Detector HPLC Solvent Reservoirs HPLC Column Rheodyne Injector 9060 Polychrom (Diode Array) Detector Computer Workstation Keep an eye on these 4 screens!
50. Varian 9010 Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure {H 2 O} {MeOH} (mL/min) (atmos.) Ready Ternary Pump A C B from solvent reservoir Column to detector to column through pulse dampener to injector through pump load inject
