High performance liquid chromatography

This presentation involves all d information about HPLC and its important components useful in academic sessions.

1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Presented by- Miss Vaishali v. dudhabale f.y. m pharm SEM II(qat) Guided by- Mr. mukesh mohite Professor, dr. d y college of pharmacy, akurdi, pune.

2. CHROMATOGRAPHY Chromatography was discovered in 1903 and was used to separated plant pigments. It is a popular method used to separate mixtures to their individual components. HPLC: The history of HPLC began in the 60’s and was known as high pressure liquid chromatography. The instrumentation and columns improved over the time and it is now known as high performance liquid chromatography. HPLC separation technique provides high speed , efficiency and high sensitivity as compared to liquid chromatography. A very small volume of sample is injected into a tube packed with a stationary phase (3-5 microns particles) and active components are separated using high efficiency columns.

3. Theory From Liquid Chromatography to High Performance Liquid Chromatography Higher degree of separation!  Refinement of packing material (3 to 10 µm) Reduction of analysis time!  Delivery of eluent by pump  Demand for special equipment that can withstand high pressures The arrival of high performance liquid chromatography !

4. Advantages of HPLC High separation capacity, enabling the batch analysis of multiple components Superior quantitative capability and reproducibility Moderate analytical conditions Unlike GC, the sample does not need to be vaporized. Generally high sensitivity Low sample consumption Easy preparative separation and purification of samples

5. Principle of HPLC Separation is based on the analyte’s relative solubility between two liquid phases. HPLC utilizes different types of stationary phase (typically, hydrophobic saturated carbon chains), a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. Analyte retention time varies depending on the temperature of the column, the ratio/composition of solvent(s) used, and the flow rate of the mobile phase. With HPLC, a pump (rather than gravity) provides the higher pressure required to propel the mobile phase and analyte through the densely packed column .

6. COMPONENTS OF HPLC 1.Solvent Reservoir : The contents of mobile phase are present in glass container. In HPLC the mobile phase or solvent is a mixture of polar and non-polar liquid components. Depending on the composition of sample, the polar and non-polar solvents will be varied. 2.Pump : pumps the mobile phase at a specific flow rate in mL/min. The pump pressure is normally between 400-600 bar. 3.Injector : Introduces the sample into the column (about 5-20 μL). 4.Column : provides separation through high pressure created by the small particles. 5.Detector : it quantifies and identify the sample components and provides information to the computer.

7. 5. Data Collection Devices or Integrator : Signals from the detector might be gathered on graph recorders or electronic integrators that fluctuate in many-sided quality and in their capacity to process, store and reprocess chromatographic information. The PC coordinates the reaction of the indicator to every part and places it into a chromatograph that is anything but difficult to interpret.

8. Factors affecting HPLC Column Parameters Column Material Deactivation Stationary Phase Coating Material Sample Parameters Concentration Matrix Solvent Effect Sample Effect Instrument Parameters Temperature Flow Signal Sample Sensitivity Detector

9. HPLC INSTRUMENT

10. Solvent reservoir and degassing unit Solvents (mobile phase) – are stored in special reservoirs connected to the pumping system – must be free of particles that can clog components & free of bubble forming gases that get trapped in column or detector Degasser Problems caused by dissolved air in the eluent Unstable delivery by pump More noise and large baseline drift in detector cell In 0rder to avoid this problems the solvent must be degassed.

11. . Three basic ways to degas solvents 1) vacuum or suction filter (0.4 or 0.2 μm ) 2) ultrasonicate (with vacuum) 3) He purge (spurges units often built in) Can purchase HPLC solvents & water - still

12. HPLC Pump The HPLC pump has to have ruggedness and at the same time should be able to provide reproducible flow characteristics run after run. The operational pressure limits have a vast range depending upon analysis requirements. In normal analytical operation the pressure can vary between 2000 – 5000 psi but in applications covered under UHPLC mode operating pressure can be as high as 15000 – 18000 psi. An ideal pump should have the following desirable characteristics Solvent compatibility and resistance to corrosion Constant flow delivery independent of back pressure

13. . Convenience of replacement of worn out parts Low dead volume for minimum problems on solvent changeover. Types of pump in HPLC: Syringe pump Constant pressure pump Reciprocating piston pump

14. Constant pressure pump Provide consistent continuous flow rate through the column with the use of pressure from a gas cylinder. Valving arrangement allows rapid refill of solvent chamber. A low pressure gas source is needed to generate high liquid pressures.

15. Syringe pump These are suitable for small bore columns. Constant flow rate is delivered to column by a motorized screw arrangement. Solvent delivery rate is set by changing voltage on the motor. These pumps deliver pulseless flow independent of column backpressure and changes in viscosity but major disadvantages are limited solvent capacity and limitation on gradient operation

16. Reciprocating piston pump Deliver solvent(s) through reciprocating motion of a piston in a hydraulic chamber. On the back stroke the solvent is sucked in and gets delivered to the column in the forward stroke. Flow rates can be set by adjusting piston displacement in each stroke. Dual and triple head pistons consist of identical piston chamber units which operate at 180  or 120  phase difference. The solvent delivery of reciprocating pump systems is smooth because while one pump is in filling cycle the other is in the delivery cycle. High pressure output is possible at constant flow rate and gradient operation is possible. However, pulse dampening is required for further elimination of pressure pulses.

17. Elution techniques Isocratic system Constant eluent composition Gradient system Varying eluent composition HPGE (High Pressure Gradient) LPGE (Low Pressure Gradient)

18. Advantages and Disadvantages of High- / Low-Pressure Gradient Systems High-pressure gradient system High gradient accuracy Complex system configuration (multiple pumps required) Low-pressure gradient system Simple system configuration Degasser required

19. HPLC Column In GC the analyte affinity for the column is influenced by temperature. In HPLC the solvent strength affects an analytes retention on column. Therefore, analogous to temp programming in GC, do solvent programming in HPLC This is also referred to as gradient elution. Gradient elution dramatically improves the efficiency of separation. Columns - usually stainless steel - can be PEEK (poly ether ether ketone) - may cost $200-$1000 packed

20. . - Length 10-30 cm, ID 4-10 mm - Packings are 3, 5, or 10 μm particle size - Most common 25 cm, 5 μ, 4.6 mm ID - N = 40,000 to 60,000 -Normally packed under 6000 psi pressure at factory as a slurry Guard columns are normally used before the analytical column to protect & increase lifetime of column – operator usually slurry or dry packs short guard column regularly with same or similar packing used in analytical column (old column material) – can purchase guard systems, cartridges, etc

21. Types of HPLC based on column type HPLC COLUMNS Normal phase HPLC Reverse phase HPLC Size exclusion HPLC Ion exchange HPLC

22. . NORMAL PHASE Column packing is polar (e.g. silica) and the mobile phase is non polar. It is used for water-sensitive compounds, geometric isomers , cis-trans isomers and chiral compounds . REVERSE PHASE The column packing is non-polar (e.g. C18) , mobile phase is water+ miscible solvent ( e.g methanol). It can be used for polar , non polar , ionizable and ionic samples .

23. . ION EXCHANGE Column packing contains ionic groups and the mobile phase is buffer. It is used to separate anions and cations . SIZE EXCLUSION Molecules diffuse into pores of a porous medium and are separated according to their relative size to the pore size. Large molecules elute first and smaller molecules elute later.

24. Sample injectors Injectors serve to introduce required sample volume accurately into the HPLC system. Sample injection into the moving mobile phase stream in HPLC is quite different from injection into a gas stream in Gas Chromatography as precise injection is required against high back pressure. In such a situation it is not possible to simply inject using a syringe alone. SAMPLE INJECTORS Manual Automatic

25. . Precautions to take while injecting sample in HPLC Prime injector with solvents to be used but it should be ensured that solvent is compatible with solvent used earlier. Needle wash between samples will prevent carry over between injections. Before start and at end of analysis ensure tubing is completely washed of buffers or previously used solvents. Do not forget to feed the vial number correctly on auto sampler rack and list out the sequence correctly in the computer.

26. Manual injector: Injection method Syringe measurement method It is desirable that no more than half the loop volume is injected. Loop measurement method It is desirable that at least 3 times the loop volume is injected.

27. Autosampler: Injection method Pressure injection method Total volume injection method

28. Column Oven Air circulation heating type Block heating type A luminum block heater Insulated column jacket type Water bath

29. HPLC Detectors . Evaporative Light Scattering Detector (ELSD) Electrochemical Mass-Spectrometric Photo-Diode Array Absorbance (UV with Filters, UV with Monochromators) IR Absorbance Fluorescence Refractive-Index

30. . 1) Filter based UV-vis detector – Typically set at 254 nm using the most prominent band in Hg spectrum – can also use 313, 365, 334 nm and other lines as well 2) Variable wavelength detectors – use continuous source like (D2 or H2) & a monochromator, select any λ, less sensitive 3) PDA - D2 or H2 source, disperse & focus on diode array, get complete spectrum every 1 sec, powerful, expensive, less sensitive, lots of data generated 4)Fluorescence detector – normally fixed wavelength filter fluorometer excitation filter & emission filter can be changed for particular λ of interest gives selectivity based on: - ability to exhibit fluorescence - excitation wavelength - emission wavelength

31. . Variable λ monochromator based fluorescence detectors also available Filter based detectors usually more sensitive 5) Refractive index detector (RI) - responds to nearly all solutes but has poor sensitivity – detects changes in refractive index as sample passes through as long as solute has different RI than solvent analogous to TCD in GC. 6)Electrochemical Detection • Amperometric – fix potential & measure current ( i ) • Conductometric – measure conductivity • Coulometric – fix potential & integrate i

32. . • Voltammetric – vary potential & measure i • Potentiometric – measure potential Can use 2 or 3 electrode design with Pt or carbon electrodes (glassy C or C paste) Electrochemical detector is nearly universal. Other HPLC detectors • LC-MS using thermospray – new popularity (pharmaceuticals) • Evaporative light scattering - polymers • LC-FTIR • LC-plasma emission or ICP-MS

33. Comparison of Detectors .

34. Evaluation parameters EFFICIENCY RESOLUTION INERTNESS RETENTION INDEX COLUMN BLEED CAPACITY FACTOR

35. Applications of HPLC The HPLC has several applications in the fields of pharmacy, forensic, environment and clinical. It also helps in the separation and purification of compound [57-83]. Pharmaceutical Applications: The pharmaceutical applications include controlling of drug stability, dissolution studies and quality control. Environmental Applications : Monitoring of pollutants and detecting components of drinking water. Forensic Applications : Analysis of textile dyes, quantification of drugs and steroids in biological samples.

36. . Food and Flavour Applications : Sugar analysis in fruit juices, detecting polycyclic compounds in vegetables, analysis of preservatives. Clinical Applications : Detecting endogenous neuropeptides, analysis of biological samples like blood and

37. REFERENCES Book of Instrumental Methods of Chemical Analysis of Gurdeep R. Chatwal & Sham K. Anand, Pg. no. 2.264.

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