HPLC

  • Created by: LBCW0502
  • Created on: 21-11-18 16:53
What can HPLC be used for?
Purification of drug, determine purity of drug, determine components in mixture, determine concentration of specific drug in mixture, determine concentration of drug/metabolite in biological fluid, drug abuse, drug PK/PD
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What is chromatography?
A separation technique based on different interactions of compounds with two phases, a mobile phase and a stationary phase as compounds travel through a supporting medium
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What are the three main components in chromatography?
Mobile phase (solvent that flows through supporting medium). Stationary phase (a layer or coating on the supporting medium that interacts with the analytes). Supporting medium (a solid surface on which the stationary phase is bound or coated)
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Give three examples of chromatography
TLC, HPLC, CC
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Describe features of TLC
A drop of a solution mixture is placed on the paper and allowed to dry. Mixture separates as solvent front advances past mixture. Rf values. Silica plate (hydrophilic). Stain plate to see spots (iodine, ninhydrin, UV, KMnO4)
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Which factor is important in chromatography?
Polarity e.g. hexene is non-polar, methanol is polar (determines retention times)
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Describe features of column chromatography
Analytes interacting most strongly with stationary phase take longer to pass through system than those with weaker interactions. Interactions usually chemical in nature but can also be physical
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Outline features of the components in HPLC instrumentation
Solvent delivery system, solvent reservoir, sample injection, column, detector, waste collection, computer/chromatogram
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Describe HPLC characteristics (1)
Columns have small diameter (1-10 mm). Columns made of stainless steel. High pressures of <6000 psi, UPLC - 18000 psi. Controlled flow of mobile phase. Precise sample introduction and small volumes
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Describe HPLC characteristics (2)
Special continuous flow detector that uses small flow rates and low detection limits. Automated sampling. Rapid analysis and high resolution
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Describe features of the stationary phase for HPLC
Particle size 1.7-10 micrometres packed tightly, pore sizes 7-30nm (from ibuprofen to proteins). SA of 5-250 m^2/g. Normal phase (SiOH), reverse phase (C8, C18, phenyl), anion exchange (NH4+), cation exchange (COO-)
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Describe features of normal phase chromatography (1)
Stationary phase (specific shape, not flat surface for SiOH). Stationary phase polar silica, mobile phase non-polar hexane. Non-polar elutes first, moderate/slow elution, polar/slower elution
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Describe features of normal phase chromatography (2)
Benzyl alcohol binds to silica, benzaldehyde binds to silica with weaker bonds, ethylbenzene doesn't bond. Elution - ethylbenzene, benzaldehyde, benzyl alcohol
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Describe features of reverse phase chromatography (1)
Silica reacts with Si(Cl3)(C8H18) to produce non-polar stationary phase (hydrophobic chains). Retention mechanism (polarity). Stationary phase non-polar/hydrophobic C18, mobile phase polar/hydrophilic (water/methanol)
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Describe features of reverse phase chromatography (2)
Non-polar analyses more attracted to non-polar stationary phase, less attracted to polar mobile phase, retained on reverse phase column. Polar analyses more attracted to polar mobile phase, less attracted to non-polar stationary phase, less retained
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Describe features of reverse phase chromatography (3)
Very polar analytes like mobile phase and elute first, moderately polar analytes like stationary phase (slow down). Non-polar analytes like stationary phase (elute last)
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Which amino acids would elute first in reverse phase chromatography?
Amino acid with more polar variable groups, followed by amino acid with moderately polar variable groups and then amino acid with non-polar variable groups
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How does the chain length of the HC in the stationary phase for reverse phase chromatography affect retention times of analytes?
More polar analytes elute first followed by non-polar analytes. Samples more retentive with larger capacity
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Describe features of gel-filtration chromatography/size-exclusion chromatography
Column filled with porous beads made of insoluble highly hydrated polymer (e.g. dextran, agarose sephadex). Large molecules flow more rapidly (small molecules get into beads).
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Describe features of ion exchange chromatography
+ charged protein binds to - charged beads and - charged proteins flow through (anion exchange). Opposite for cation exchange (negatively charged proteins bind to + charged beads, + charged proteins flow through). E.g. NH4+ and COO-
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How do ions in the ion exchange column get removed?
Mixture of proteins in Mes buffer loaded into cation exchanger. + proteins adsorb to media/displace Na cations (pH can be adjusted). CM/DEAE groups bind to cellulose/agarose
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State components on a chromatogram
Retention time (tr), void time (tm), baseline width of peak in time units (wb), half height width of peak in time units
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How do you calculate the capacity factor?
k = retention time - void time / void time
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What is the separation factor?
Alpha - how well two solutes are separated by the chromatographic system
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How do you calculate the separation factor?
Alpha = k'2/k'1 (k'1 is the capacity factor of the first solute, k'2 is the capacity factor for the second solute). Alpha values >1.1 indicates good separation
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Describe features of resolution
Resolution between two peaks is the second measure of how well peaks are separated. Rs (preferred/both retention time and column efficiency considered, Rs = 1.5 represents baseline resolution/complete separation of two solutes, Rs = 1.0 adequate)
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How do you calculate Rs?
Rs = tr2 - tr1 / wb2 + wb1
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Describe features of the plate theory (1)
View column as divided into N of adjacent segments (theoretical plates). Within each theoretical plate, analytes completely equilibrate between stationary phase and mobile phase
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Describe features of the plate theory (2)
Height equivalent of a theoretical plate (H) compares efficiency of columns with different lengths (L = length of column, N = number of theoretical plates). Larger N, lower H (more efficient). H = L/N
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Describe features of the plate theory (3)
H can be used to relate chromatographic parameters, flow rate, particle size to kinetic parameters resulting in peak broadening
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How do you calculate the number of theoretical plates (N)?
N = 16 x (tr)^2/w^2. Alternative way: N = 5.55 x (tr)^2/(w 1.5)^2. To allow for peak tailing - calculate using equation with peak width at half height
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What are the issues with the plate model?
Fails to relate band broadening process to experimental parameters e.g. particle size, stationary phase film thickness and mobile phase viscosity
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Describe features for rate theory of chromatography (graph)
u = linear velocity (flow rate x Vm/L). H = total plate height of column. A = constant represents eddy diffusion and mobile phase mass. B = constant representing longitudinal diffusion. C = mobile phase and stationary phase mass transfer
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Describe features of Eddy diffusion (term A)
A process that leads to peak broadening due to the presence of multiple flow paths through a packed column. Some solute molecules arrive at end sooner due to different path around support particles
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Describe features of longitudinal diffusion (term B) - 1
Analyte within mobile phase travels through column, undergoes diffusion into surrounding region (3 geometrical axes). Occurs along length of column.
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Describe features of longitudinal diffusion (term B) - 2
Longitudinal liquid diffusion diminished at high temperatures. Effect of pressure on longitudinal diffusion insignificant due to liquids being incompressible
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Describe features of mobile phase mass transfer (1)
Process of peak broadening caused by presence of different flow profile within channels or between particles of support in column. Solute in centre of channel moves faster than solute at edges, reaches end first (band broadening)
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Describe features of mobile phase mass transfer (2)
Degree of band-broadening due to eddy diffusion and mobile phase mass transfer depends mainly on: size of packaging material and diffusion rate of solute
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Describe features of stagnant mobile phase mass transfer (1)
Band broadening due to differences in diffusion rate of solute between mobile phase outside pores of support (flowing mobile phase) to mobile phase within pores of support. Solute doesn't travel down column, spends longer in column
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Describe features of stagnant mobile phase mass transfer (2)
Degree of band-broadening due to stagnant mobile phase mass transfer depends on: size/shape/pore structure of packaging material, diffusion/retention of solute, flow-rate of solute through column
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Describe features of the Van Deemter equation (1)
Tries to account for all kinetic processes occurring during separation. Lambda (packing factor, 0.5-1.0 depend on column packing). dp (particle diameter, size of particle/distribution, smaller/regular sized particles, smaller lamba)
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Describe features of the Van Deemter equation (2)
Dm (diffusion coefficient of mobile phase, accounts for Cs term/mass transfer in stationary phase mass transfer), u (mean linear velocity), gamma (irregularity of pores)
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Compare isocratic elution and gradient elution
Isocratic elution (constant concentration of mobile phase over time e.g. concentration of methanol, leads to peaks being very close together). Gradient elution (change percentage of methanol over time, achieve longer retention times/separated peaks)
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What are the details for an analytic columns?
Internal diameters of 4.6 mm, capacity of 1 mg, flow rate of 1.0 mL/min (values specific to instrumentation - new equipment/sensitivity improvement, HPLC, UPLC)
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Outline the HPLC instrumentation overview
Principle pattern - solvents, pumps, mixer, injector, column, detector, waste collection. Example - solvent reservoirs in cabinet, controlled, vacuum degasser, binary pump, autosampler, thermostate, column, detector
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Describe features of solvent filters (1)
Solvent inlet filter - stainless steel/glass with 10 micron porosity, removes particulates from solvent
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Describe features of solvent filters (2)
Pre-column filter - used between injector and guard column (2-0.5 micron), remove particulates from sample/autosampler wear debris (must be well designed to prevent dispersion)
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Describe features of vacuum degassing
Solvent pass through tubular plastic membrane in vacuum container with pump. Passes through sensor/control units. Use of vacuum pump. Removes air from system (air could interfere with results in sample)
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Describe features of the solvent delivery system
Provide accurate/constant flow, provide accurate mobile phase compositions, provide force necessary to push mobile phase through tightly packed column. Pump is the most critical piece of equipment for a successfully operating HPLC
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What are the performance parameters for HPLC pumps?
Flow precision, flow range, delay volume, pressure pulse, composition precision
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Describe features of sample injection
Reproducible introduction of sample volume into mobile phase flow. Two major designs - automatic injectors or manual injectors (load-inject, sample loop, use of pump to get sample to column and collection of waste)
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What are the types of HPLC detector?
UV-Vis (diode ray, multiple wavelength, variable wavelength). MS, refractive index, fluorescence, light scattering, electrochemical, radioactivity, conductivity. (Fluorescence has better sensitivity then UV)
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What information can be produced from peaks in a chromatogram by UV and MS detectors?
UV - peak spectrum shows which functional groups are present. MS - peak spectrum gives information about the structure of the molecule with fragmentation patterns - (use of qualitative information)
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What are the detector performance characteristics?
Sensitivity (LoD, LoQ), selectivity, linearity, qualitative information, reliability, ease of use and universality
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Describe features of the UV-Vis detector
Fraction of light transmitted through detector cell is related to solute concentration according to Beer's Law. Characteristics - specific, concentration sensitive, good stability, gradient capability. UV-Vis spectral capability (diode array tech)
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Describe features of detector design
Variable wavelength detector. Single wavelength detection of multi wavelength detection possible. Wavelength calibration done automatically using holmium filter
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Card 2

Front

What is chromatography?

Back

A separation technique based on different interactions of compounds with two phases, a mobile phase and a stationary phase as compounds travel through a supporting medium

Card 3

Front

What are the three main components in chromatography?

Back

Preview of the front of card 3

Card 4

Front

Give three examples of chromatography

Back

Preview of the front of card 4

Card 5

Front

Describe features of TLC

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