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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Mobile phase selection Column selection Efficiency parameter Resolution Presented by, Amrutha Felix M. Pharm (Pharmaceutics) 1

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Liquid chromatography in which the mobile phase is forced through the column at a high rate using pumps known as HPLC pumps. Two types: Normal Phase Chromatography Reverse Phase Chromatography 2


MOBILE PHASE Consists of mixture of organic solvents or aqueous organic solvents or buffered solution depending on analytical method along with additives. Stored in solvent reservoir. 3

Important factors:

Important factors Should not degrade equipment or column packing – strong acids, bases, halide solutions avoided. Chemically pure – spectro or HPLC grade solvents recommended. Volatility to be considered in case of sample recovery. Viscosity should be less than 0.5 centipoise . Should be degassed to give reproducible retention times for eluting peaks. 4

Mobile phase selection :

Mobile phase selection Selected by matching the relative polarity of the solvent to that of the sample components. First, a solvent is chosen to match most of the functional group in the sample. E.g. alcohols for hydroxyl group, ketone or acetate for carbonyl group If the capacity factor (k’) values are too small (sample elutes too rapidly), then a weaker (less polar) solvent is substituted. Conversely, if a sample does not elute in a reasonable time because of high k values, then a solvent with high polarity is selected. 5

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Based on HPLC Normal phase : non polar solvents (hexane, diethyl ether) Reverse phase : mixture of water and some polar organic solvents such as acetonitrile and methanol. Based on elution Isocratic elution : mobile phase composition is constant. Gradient elution : mobile phase composition is changed during separation. Based on elution strength High polar solvent decreases retention. Less polar solvent increases retention. 6


COLUMN SELECTION Depending on use Analytical column Preparative column Depending on nature Open tubular/ golay column SCOT Column (Support Coated Open Tubular Columns) WCOT Column (Wall Coated Open Tubular Columns) Packed Column 7

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Packed Columns Fabricated from glass or metal tubing. Length : 2-3m, internal diameter :2-4mm. Advantages No absorption of polar compounds. Sample overload recovery is rapid. Retention data is highly reproducible. 8

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Main part of HPLC Properties Should withstand the operating pressure. Materials should not corrode. Non reactive with mobile phase and solute. Dimensions of column External diameter : 6 – 10mm Internal diameter : 4 – 8 mm Length : 10 – 40 cm Particle size: 5 – 10 microns 9

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A typical column should show: Satisfactory peak shapes (minimal tailing) Satisfactory peak width (narrower) Stable detector base line Good resolving power The column is selected based on the following: Attainable resolution Speed of analysis Load capacity of column Polarity of sample 10

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Column diameter Defines how much sample can be introduced into column. Column type Diameter Sample load Analytical 4.6 mm 0.1 -1.5 mg Preparative 21.2 mm 20 -250 mg Semi preparative 9.4 mm 1 -10 mg 11

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Flow rate It depends on the diameter of the column Column type Diameter Flow rate Analytical 4.6 mm 0.5 – 3ml/min Preparative 21.2 mm 20 – 60ml/min Semi preparative 9.4 mm 5 – 10ml/min 12

Factors affecting column efficiency :

Factors affecting column efficiency Particle size Efficiency of column depends on size of the particles used in stationary phase. As the particle size decreases, the exposed surface area of the stationary phase increases. As a result retention time, retention volume and column efficiency increases. Viscosity of solvents As viscosity of mobile phase increases column efficiency increases. Temperature of column As temperature is increased elution rate increases but adsorbent power of the column decreases. 13


STATIONARY PHASE Less volatile. Thermally stable. Chemically inert. The liquid phase is generally grouped into: Non-polar : paraffin oils like neugol , silicon oils like sqaulene . Intermediate polar : possess polar or polarisable group attached to a non polar skeleton. Polar : poly glycols, carbowaxes Liquid having hydrogen bonding : glycerol 14

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Liquid stationary phases are either coated on the particles or are chemically bonded to it. The different types of particles used in HPLC are: Microporous particles Perfusion packings Nonporous packings Chiral stationary phases Microbore columns 15


EFFICIENCY PARAMETERS Two important parameters include: Plate height (H) Plate count (N) N = L/H L- Length of column packing PLATE THEORY It states that ‘ a theoretical plate is an imaginary or hypothetical unit of a column where distribution of solute occurs between stationary phase and mobile phase.’ 16

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Plate height – distance a solute moves while undergoing a partition. H= L/N eff N eff = ( t R / σ ) 2 N eff = effective plate number 17

Height equivalent to theoretical plate (HETP):

Height equivalent to theoretical plate (HETP) A theoretical plate can be of any height which decides the efficiency of separation. If HETP is less, the column is more efficient. HETP = length of column no of theoretical plates HETP is given by Van Deemter equation; HETP = A+ B + C µ µ 18

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A = Eddy diffusion term B = Longitudinal diffusion term C = Effect of mass transfer which depends on flow rate µ = Velocity of mobile phase 19

Retention Time (Rt):

Retention Time ( R t ) Difference in time between the point of injection and appearance of peak maxima Time required for 50% of a component to be eluted from a column. Measured in minutes or seconds. Proportional to the distance moved on a chart paper, which can be measured in cm or mm. 20

Retention Volume ( VR):

Retention Volume ( V R ) Volume of mobile phase necessary to convey a solute band from the point of injection through the column, and to the detector. V R = R t × F F = average flow rate of solute within column 21

Capacity factor/Retention factor (k’):

Capacity factor/Retention factor (k’) Degree of retention of the sample component in the column. It is defined as the time the solute resides in the stationary phase ( R t ) relative to the time it resides in the mobile phase (t 0 ). k’ = R t t 0 Used to compare the retention of a solute between two chromatograms. For an analyte ideal retention factor is 1-5. K’ <1, elution is fast, determination of retention time is difficult. 22

Separation/selectivity factor (α):

Separation/selectivity factor ( α ) Ratio of capacity factors of both peaks or the ratio of its adjusted retention times Values measured from a chromatogram containing two peaks. α = t R1 - t o / t R2 - t o α = k’1/k’2 23


RESOLUTION(R s ) Measure of degree of separation of two peaks of adjacent analytes . R s = √N ( α -1) (1+k’) 4 α k’ 24

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Larger resolution, better separation. R s = 0; no separation R s = 0.6; slight partial separation R s = 1; partial separation R s = 1.5; baseline separation 25


REFERENCES 26 Instrumental methods of analysis, 7 th edition, Willard, Page No: 593-597. Instrumental methods of chemical analysis, Gurdeep R. Chatwal , Page No:2.631. Pharmaceutical analysis II-instrumental methods, P.C.Kamboj , Page No: 77-92. Textbook of pharmaceutical analysis, 3 rd edition, Dr. S. Ravisankar , Page No: 18.1 – 18.15

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