hplc aruna
TRANSCRIPT
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HPLC
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HPLC
originally referred to:
High Pressure Liquid Chromatography
high pressure to be able to use small particle size to allowproper separation at reasonable flow rates
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PRINCIPLE
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Principle of HPLC:The principle of HPLC are based on Van Deemter equation which
relates the efficiency of the chromatographic column to theparticle size of the column, molecular diffusion and thickness ofstationary phase.
The Van Deemter Equation is given as
H or HETP = A + B + C
where, A represents eddy diffusion
B represents molecular diffusionC represents rate of mass transfer
represents flow rate
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Basic Components of an HPLC
System1.Pump System.Mobile phase pressures up to 6000 psi are necessary to
achieve reasonable column elution times (~ minutes). Typical flowrates are 0.1 to 10 mL/minute.
2.Injection System.Used to introduce small samples (0.1 to 500 L) intothe carrier stream under high pressure.
3.Reservoirs (Solvents).Multiple solvents are necessary for performinggradient elution's (i.e. changing the polarity of the mobile phaseduring a run).
4.Chromatographic Column. Typically 10-30 cm in length containing apacking of 5-10 m diameter. Many types of columns are available,depending on the type of liquid chromatography desired.
5.Detector.Many types are available including UV, IR, refractive index,fluorescence, conductivity, mass spectrometry, and electrochemical.Diode array detectors are used when wavelength scans are desired.
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HPLC Basic Instrumentation
Mobile
phasePump
Solvent Delivery
Injector
Sample Injection
Column
Separation
Detector
Data Processor
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HPLC system
Solvent Reservoir
Degasser
Solvent Delivery System (Pump)
Injector
Column &oven
Detectors
Recorder (Data Collection)
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PUMP SYSTEM
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HPLC PumpHPLC Pump
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Solvent Reservoir
Usually one or more glass or stainless steel reservoirs each
of which contains 200-1000 ml of solvent
Isocratic elution - single solvent separation technique Gradient elution - 2 or more solvents, varied during
separation
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SAMPLE INJECTION SYSTEM
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HPLC Autosampler and InjectorHPLC Autosampler and Injector
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from Pump from Pump to Column
Vial
Needle
Measuring Pump
to Column
LOADLOAD INJECTINJECT
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HPLC ColumnHPLC Column
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HPLC DetectorHPLC Detector
UV/Visible SpectrophotometerUV/Visible Spectrophotometer
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Column
straight, 15 to 150cm in length; 2 to 3mm i.d.
packing - silica gel,alumina, Celite
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CHARACTERISTICS OD
DETECTORS Have high sensitivity and the same predictable response Respond to all solutes, or else have predictable specificity Have a wide range of linearity Be unaffected by changes in temperature and mobile-phase
flow
Respond independently of the mobile phase Not contribute to extra column band broadening Be reliable and convenient to use Have a response that increases linearly with the amount of
solute Be nondestructive of the solute Provide qualitative information on the detected peak Have a fast response
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HPLC
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Degasser
Problems caused by dissolved air(O2, N2)in mobile phase
Unstable delivery in pump
Bigger noise and large baseline-drift in detector cell
In order to avoid causing the problems,
mobile phase should be degassed.
vacuum pumping systems
distillation system
a system for heating and stirring the solvents
sparging system - bubbles an inert gas of low solubility through the
solvent
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Solvent Delivery System
Requirements
ability to mix solvents and vary polarity of mobile phase during
run
unlimited solvent reservoir
generation of pressures up to 6000 psiflow rates ranging from 0.1 to 10 mL/min
flow reproducibilitys of 0.5 % or better
resistance to corrosion by a variety of solvents
pulse-free output
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Solvent Delivery System
RheodyneInjector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
Ternary Pump
A
C
B
from solventreservoir
Co
lum
n
todetector
to column
through
pulse
dampener
to injector
through pump
load
inject
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Chromatography II: HPLCHPLC Waste CollectionHPLC Waste Collection
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Use a 10 mL volumetric pipet to add 10.00 mL of soft
drink to 10.00 mL of deionized water.Mix thoroughly and half fill a HPLC vial with
your sample. Label the vial with your name and thename of the soft drink.
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Analysis of ResultsUse the four caffeine standards to prepare a
calibration curve (graph) that plots peak area vs.
concentration.
Draw a best fit straight line on your graph.Use your calibration curve to determine the
concentration of caffeine in the sample you prepared.
Use the concentration of caffeine in your sample,
along with the dilution equation, to determine theconcentration of caffeine in the soft drink you used.
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HPLC Chromatogram of Standard 1HPLC Chromatogram of Standard 1
0.500 x 100.500 x 10-4-4 M caffeineM caffeine
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HPLC Chromatogram of Standard 2HPLC Chromatogram of Standard 2
1.00 x 101.00 x 10-4-4 M caffeineM caffeine
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Calculations
Remember, the original soft drink was diluted to prepare your HPLCsample. The concentration (M) of caffeine in the original soft drink can
be calculated by using the dilution equation:M1V1 = M2V2
V2
M1
V1M2 =
The concentration (mg caffeine / 500 mL soda) of caffeine in a full can
of your soft drink can be determine using the following equation:
M2 x 194.2 g caffeine x 1000 mg x 0.500 L = mg caffeine
mol caffeine g in bottle
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HPLC Applications
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HPLC - Applications
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