HPLC

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9/4/2011 1 High Performance Liquid Chromatography GIRISH SUKRE ,MOHSEEN SHAIKH NILESH TRIBHUVAN, ABHIJEET BAGAL AISSMS COLLEGE OF PHARMACY, PUNE, INDIA

Points : 

Points Introduction Syllabus points Principle Various types in practice Instrumentation Application Summary 9/4/2011 2

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Principle Instrumentation Pumps:- (Reciprocating pumps, Displacement pumps, pneumatic pumps), Mobile phase reservoirs, Solvent treatment systems, isocratic elution, gradient elution, Injection system. Detectors:- Photometric detectors, (single wavelength, multi wavelength, variable wavelength, programmable, diode array, fluorescence detectors), Refractive Index detectors, electrochemical detectors. Columns:- analytical column, guard column, Column thermostat, Types of column packing. 9/4/2011 3 Syllabus points

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High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, is essentially a form of column chromatography in which the stationary phase consists of small particle packings (3-50 µm) contained in a column with a small bore (2-5 mm), one end of which is attached to a source of pressurised liquid eluant (mobile phase). The three forms of high performance liquid chromatography most often used are ion-exchange, partition and adsorption. 9/4/2011 4 HPLC:- Definition (as per I.P.)

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The origins of Liquid Chromatography began in the early 1900’s with the work of the Russian botanist, Mikhail S. Tswett. His famous studies focused on separating compounds (leaf pigments), which were extracted from plants using a solvent. 9/4/2011 5 History

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Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. Like open-column chromatography, paper chromatography, and thin-layer chromatography. High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. 9/4/2011 6

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9/4/2011 7 A+B+C A+B A A+B+C Frontal analysis A A+B A+B+C

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9/4/2011 8 C A+B A D Volume of the solvent Relative conc. A B C D Displacement Anaysis B C +D B+C D

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9/4/2011 9 Conventional Column Chromatography or Open column chromatography

Disadvantages : 

Disadvantages Mobile phase moves under gravity feed or low pressure pumping Slow flow rates. Separation times are higher , even in hrs. Sample is to be collected separately & then passed on to the detector. No good efficiency. Not very reproducible.

Advantages of HPLC over Conventional column Chromatography : 

Advantages of HPLC over Conventional column Chromatography Provides a specific sensitive and precise method for analysis of complicated samples. Ease of sample preparation & introduction. Speed of analysis Analysis is specific, accurate & precise. Substances that are thermolabile, or have low volatility could be analysed by HPLC, unlike GC.

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9/4/2011 12 Principle

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Normal phase HPLC 9/4/2011 13 Various types in use

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Reversed phase HPLC 9/4/2011 14 Ion exchange HPLC Size exclusion/ Gel HPLC Bioaffinity HPLC/ Chiral Chromatography

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9/4/2011 15 Instrumentation

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Isocratic elution & Gradient elution : 

Isocratic elution & Gradient elution There are 2 basic types of chromatographic mobile phase operating modes which can be used in HPLC. The first type is called “Isocratic”. In this mode, the mobile phase composition remains the SAME throughout the run. The second type is called “Gradient” chromatography. This type is used for very complex samples with many compounds. In this mode, the mobile phase composition is CHANGED during the separation. As the separation proceeds, the elution strength of the mobile phase is strengthened.. 9/4/2011 17

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Gradient elution can be carried out by two ways Use a system where there are two bottles of solvents and two pumps. The speed of each pump is controlled (Gradient Controller) over time to deliver more or less of each solvent. The two streams are mixed (Mixer) to create the mobile phase gradient over time. At the beginning, the mobile phase is made up of mostly the weaker solvent. As the time progresses, the proportion of the stronger solvent goes up Another system design uses a single pump and multiple solvent bottles. The flow from each bottle is controlled by a gradient proportioning valve which creates the desired solvent strength 9/4/2011 18

i] Mobile Phase Reservoir & solvent treatment system : 

i] Mobile Phase Reservoir & solvent treatment system Reservoirs are made up of stainless steel. Solvent treatment involves degassing & mixing them in proper proportions if required. Degassing can be done by following different methods Spraying (on-line) Heat (off-line) Vacuum (off-line) Sonication (off-line) Distillation (off-line) 9/4/2011 19

ii] Pumps : 

ii] Pumps Required to deliver a constant flow of mobile phase at a pressure ranging from 1 to 550 bar ( 0.1-55 MPa; 14.6-8000 psi) Those with very high pressure provide wide range of flow rate Low flow rates(10-100 microlit/min.)  microbore columns, Intermediate flow(0.5-2 ml/min.)  conventional analytical HPLC columns, High flow rates  preparative or Semi preparative columns & for slurry packing techniques. 9/4/2011 20

Criteria for selection of pump : 

Criteria for selection of pump Reproducible mobile phase flow rate. Baseline pulsation should be minimum To minimize detector noise for trace analysis. Should be suitable for operation at various pressures (3000-6000 psi). Provide constant flow rate. Made up of materials resistant or chemically inert to the chemical & solvents commonly used. Adaptable to gradient analysis. Suitable for use of small volumes of solvents. 9/4/2011 21

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Protect from Evaporation. Low maintainance cost. Suitable for operation at variable flow rates Analytical : 0.5 to 2ml/min. Preparatie : 0 to 10ml/min. Mcrobore columns: microlit/min. Should have small hold up volume. 9/4/2011 22

Types of pumps : 

Types of pumps Syringe type or screw driven pumps e.g. Displacement pump. Reciprocating pumps Single piston pump Dual piston pump Reciprocating diaphragm pump. Pneumatic pumps Direct pressure pumps Amplifier pumps Constant pressure pumps 9/4/2011 23

1. Syringe type / Displacement pump. : 

1. Syringe type / Displacement pump. 9/4/2011 24

ADVANTAGES DISADVANTAGES : 

ADVANTAGES DISADVANTAGES Pulse free delivery at high pressure Independent of viscosity of solvent Limited solvent capacity Change of solvent is inconvinient Suitable for batch operation & no continuous operation possible. 9/4/2011 25

2.Reciprocating pump : 

2.Reciprocating pump 9/4/2011 26

ADVANTAGES DISADVANTAGES : 

ADVANTAGES DISADVANTAGES Constant flow rates Independent of viscosity of solvent Suitable for continuous operation. Gradient elution possible Relatively inexpensive Wide range of flow rates achieved. Pulsating output. 9/4/2011 27

3. Pneumatic pumps : 

3. Pneumatic pumps ADVANTAGES Inexpensive Easy to operate Pulse free DISADVANTAGES Flow rate dependant on viscosity of solvent. Gradient operation is difficult. 9/4/2011 28

4. Constant Pressure Pump : 

4. Constant Pressure Pump 9/4/2011 29

4. Constant pressure pumps : 

4. Constant pressure pumps ADVANTAGES High liquid pressure achieved Valving arrangement provide rapid refill of solvent chamber (capacity 70ml) Pulse less & continuous pumping DISADVANTAGES Inconvenient for gradient analysis. 9/4/2011 30

iii] Injectors : 

iii] Injectors The samples should be introduced with high degree of reproducibility for better efficiency. Overloading  band broadning Sample injection should not disturb the pressure system. 9/4/2011 31

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Sample injectors/ Injection ports are of 3 basic types Syringe injection: - sample is injected directly into the column through a self sealing septum with a syringe that can withstand pressure up to 1500 psi. Stop flow injectors: - injected with syringe only but by removing upper head of the column after stopping the solvent flow momentarily. Solvent flow injectors: - sample is deposited before the column inlet & then swept by a valving action into the column by the mobile phase. i.e. injection valves. This does not disturb pressure system at all. 9/4/2011 32

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9/4/2011 33 Overloaded Injection :

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HPLC injection valve (Rheodyne Injection) : 

HPLC injection valve (Rheodyne Injection) 9/4/2011 35 LOAD INJECT

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iv]Columns : 

iv]Columns The columns are made up of high quality stainless steel polished internally to mirror finish. Smaller diameter & longer length of column ensures better efficiency, as it increases no. of theoretical plate but shorter length provides faster separation. 9/4/2011 37

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Types of columns Analytical columns (length 25-100 cm in & internal diameter 2-6 mm) Preparative columns (length 25-100 cm in & internal diameter 6 mm or more ) Guard columns protective, have large diameter packings. Column thermostat:- For certain applications, close control of column temperature is required. Better chromatograms are by maintaining column temp. constant to a few tenths degree centigrade. Generally columns have heaters that maintain temperature between 100-150ْ C. Columns may also be fitted with water jackets fed from a constant temp. bath to give precise temp. control. 9/4/2011 39

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Types of column packings:- Pellicular Porous particle The material varies with the type of interaction involved in separation. Adsorption HPLC e.g. HPLC grade silica Partition HPLC i] Normal phase HPLC e.g., ethane 1-2 diole (polar) & ii] Reversed phase HPLC e.g., Octadecylsilane (non-polar) Ion exchange HPLC e.g., ion exchange residues chemically bonded with silica. Size exclusion HPLC e.g., sephadex 9/4/2011 40

v]Detectors : 

v]Detectors Classification Photometric detectors Single wavelength detectors Multiwavelength detector Variable wavelength detector Programmable detector Diode array detector Fluorescence detector Refractive index detectors Mass spectrometry (MS) Other detectors Electrochemical (ECD), conductivity, infrared, mass (evaporative), radioactivity and post-column reaction system 9/4/2011 41

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9/4/2011 42 UV/Vis Absorbance Detector: Schematic

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9/4/2011 43 Characteristics of a UV/Vis Detector UV/Vis absorbance detector typically consists of : A deuterium source (190 - 700 nm) A monochromator involving a moveable grating controlled by stepper motor to select a certain wavelength through the exit slit to a small flow cell (about 10 mL) Two photodiodes to measure the light intensity of the sample and reference beam Principle for absorbance detector is the Beer’s Law Absorbance = molar absorptivity x pathlength x concentration A = e b c = - Log I / I0 where I0 = Initial light intensity It is the most common detector for HPLC, capable of ng detection Noise/drift characteristics important for sensitivity

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9/4/2011 44 The most powerful UV/Vis absorbance detectors in use today are photodiode-array (PDA) based instruments It permit that very rapid collection of data over a selected spectral range. Thus, spectral data for each chromatographic peak can be collected and stored. This stored data may then be compared with the spectrum of a pure standard from a library - a spectral analysis study of peak purity. The PDA detector is very useful for the identification of components that are difficult to resolve (overlapping peaks) since the characteristic spectrum for each of the unresolved components is likely to be different. PDA Detectors

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9/4/2011 45 Photodiode Array Detector: Principles Principles and components similar to that of UV/Vis detector A flow cell is placed before a stationary grating and passes the entire light spectrum of the light source A diode array with many elements measures the spectral intensity of each wavelength Records spectra to assist the determination of peak identity and purity

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9/4/2011 46 Photodiode Array Detector: Schematic

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Types Bulk property detectors: e.g., refractive index detectors. Solute property detectors: e.g., Photometric detectors & fluorescence detectors. 9/4/2011 47

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Detection : 

Detection 9/4/2011 49

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Preparative HPLC Chemical Separations Purification Identification Quantification 9/4/2011 51 Application

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9/4/2011 52 Sterile exclusion , aqueous mobile phase Sterile exclusion , non-aqueous mobile phase Water soluble MW<2000 Sample MW<2000 Nonionic Ionic Sterile exclusion , aqueous mobile phase Sterile exclusion , aqueous mobile phase Anion exchange or ion pair Cataion exchange or ion pair Acidic Basic Water insoluble Low polarity Medium high polarity

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9/4/2011 53 THANK YOU

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9/4/2011 54 Any Questions?