Hplc

About HPLC, Terminologies, instrumentation and validation

1. HPLC Ahsan Saqi The Islamia University of Bahawalpur

2. High Performance Liquid Chromatography HPLC is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution

3. Chromato-graphy / -graph / -gram / - grapher Chromatography: Analytical technique Chromatograph: Instrument Chromatogram: Obtained “picture” Chromatographer: Person

4. Absorption: In chromatography, absorption signifies the process by which a solute partitions into a liquid-like stationary phase. Adsorption: The process by which a chemical entity is accumulated on a surface. Mobile Phase: The eluate moving through the column. In gas chromatography (GC) this will be a gas, and in liquid chromatography (LC) a liquid. Stationary Phase: The substance that remains in one place in the column. In GC this will be a liquid of high-viscosity, which clings to the inner walls of the column; in LC it will be some sort of packing, either solid or gel-based .

5. Capillary Column: A column whose inner diameter is under 0.5 mm. Eluate: The mobile phase exiting a column. Eluent: The mobile phase entering a column. Elution: The passage of the mobile phase through the column to transport solutes

6. Partition Chromatography: A type of chromatography based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. Flow Rate: The amount of mobile phase that has passed through the column per unit time. The units are milliliters per second (mL/sec) or, more commonly, milliliters per minute (mL/min). Solute: The term for the sample components being analyzed.

7. Instrumentation

8. HPLC System Solvent Delivery System Variable UV/Vis Detector HPLC Solvent Reservoirs HPLC Column Injector Detector Computer Workstation Keep an eye on these 4 screens!

9. Solvent Delivery System

10. Solvent Delivery System 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

11. UV/ Visble Detector ABS Flow rate l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready

12. HPLC Working

13. Pump Sample injection unit (injector) Column Column oven (thermostatic column chamber) Detector Eluent (mobile phase) Drain Data processor Degasser Flow Channel Diagram for High Performance Liquid Chromatograph

14. Picture of an HPLC column

15. HPLC Column Material Stainless steel (SUS) PEEK (polyether ether ketone) Fluororesin O.D. (outer diameter) 1.6 mm I.D. (inner diameter) 0.1 mm 0.3 mm 0.5 mm 0.8 mm etc.

16. Column Storage Storage Solution It is generally safe to use the same storage solution as used at shipment. In order to prevent putrefaction, alcohol or some other preservative substance may be added. Storage Conditions Insert an airtight stopper in the column end. Never allow the packing material to dry. Make a record of the storage solution and final usage conditions and store it together with the column. Store the column in a location not subject to shocks or sudden temperature changes.

17. WHAT will AFFECTS the SYSTEM? Column Parameters Column Material Deactivation Stationary Phase Coating Material Instrument Parameters Temperature Flow Signal Sample Sensitivity Detector Sample Parameters Concentration Matrix Solvent Effect Sample Effect

18. HPLC Chromatogram

19. HPLC Chromatogram t R t Intensity of detector signal Time Peak t R : Retention time h A t : Non-retention time A : Peak area h : Peak height

20. HPLC Chromatograms R t = 3.0 min. faster moving less retained R t = 5.2 min. slower moving more retained 0 1 2 3 4 5 6 7 Time (minutes) Absorbance  Approximation of peak area by triangulation Area = base x height 2 base height Peak A Peak B

21. Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

22. Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

23. Normal vs. Reversed Phase Chromatography

24. Chromatography Stationary Phases relatively polar surface bulk (SiO 2 ) x surface relatively nonpolar surface Silica Gel bulk (SiO 2 ) x surface Derivatized Silica Gel Where R = C 18 H 37 hydrocarbon chain (octadecylsilyl deriv. silica or “C18”) “normal phase” “reversed phase”

25. Validation

26. Validation: A programe which guarantees the accuracy, specificity, precision and robustness of a method or process.

27. Validation of method Scientifically demonstrating that the analytical methods concur with the intended purpose (i.e., that errors are within a permissible range) Evaluating required items from the validation characteristics Validation characteristics Accuracy / trueness Precision Specificity Detection limit Quantitation limit Linearity Range (Robustness)

28. Accuracy / Trueness Definition Degree of bias in measurements obtained with analytical procedures Difference between true value and grand mean of measurements Evaluation Method Comparison with theoretical values (or authenticated values) Comparison with results obtained using other analytical procedures for which the accuracy (trueness) is known Recovery test

29. Precision Definition Degree of coincidence of series of measurements obtained by repeatedly analyzing multiple samples taken from a homogenous test substance Variance, standard deviation, or relative standard deviation of measurements Repeatability / Intra-Assay Precision Precision of measurements taken over a short time period under the same conditions Intermediate Precision Reproducibility

30. Specificity Definition The ability to accurately analyze the target substance in the presence of other expected substances The discrimination capability of the analytical methods Multiple analytical procedures may be combined in order to attain the required level of discrimination Evaluation Method Confirmation that the target substance can be discriminated (separated) from co-existing components, related substances, decomposition products, etc. If reference standards for impurities cannot be obtained, the measurement results for samples thought to contain the impurities are compared .

31. Detection Limit Definition The minimum quantity of a target substance that can be detected. Quantitation is not absolutely necessary . Evaluation Method Calculated from the standard deviation of measurements and the slope of the calibration curve. DL = 3.3  / slope (  : Standard deviation of measurements) (Slope: Slope of calibration curve) Calculated from the signal-to-noise ratio. Concentration for which S/N = 3 or 2

32. Quantitation Limit Definition The minimum quantity of a target substance that can be quantified Quantitation with an appropriate level of accuracy and precision must be possible. (In general, the relative standard deviation must not exceed 10%.) Evaluation Method Calculated from the standard deviation of measurements and the slope of the calibration curve. QL = 10  / slope (  : Standard deviation of measurements) (Slope: Slope of calibration curve) Calculated from the signal-to-noise ratio. Concentration for which S/N = 10

33. Linearity Definition The ability of the analytical method to produce measurements for the quantity of a target substance that satisfy a linear relationship. Values produced by converting quantities or measurements of the target substance using a precisely defined formula may be used. Evaluation Method Samples containing different quantities of the target substance (usually 5 concentrations) are analyzed repeatedly, and regression equations and correlation coefficients are obtained. Residuals obtained from the regression equations of the measurements are plotted, and it is confirmed that there is no specific slope.

34. Range Definition The region between the lower and upper limits of the quantity of a target substance that gives appropriate levels of accuracy and precision Evaluation Method The accuracy, precision, and linearity are investigated for samples containing quantities of a target substance that correspond to the lower limit, upper limit, and approximate center of the range.

35. Robustness Definition The ability of an analytical procedure to remain unaffected by small changes in analytical conditions. Evaluation Method Some or all of the variable factors (i.e., the analytical conditions) are changed and the effects are evaluated.

36. Advantages of methods validation: There are a number of very good reasons why analytical methods are validated. First, the laboratory conducting the tests will have an increased output with fewer reductions in rejections of the results. The method is therefore cost effective and avoids additional capital expenditure on other equipment and reduced testing of the finished goods e.g. a batch of drugs.