Dipaks PPT HPLC fundamentals

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SCHOOL OF CHEMICAL SCIENCES NORTH
MAHARASHTRA UNIVERSITY JALGAONPresented
byGOSAVI DIPAK PRAKASH
Fundamentals of Liquid Chromatography (HPLC)
Agilent 1100 Series HPLC Value System and
chemstation online
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High Performance Liquid Chromatography (HPLC)

• HPLC  is now one of the most powerful tools in
  analytical chemistry.
• It has the ability to separate, identify, and
  quantitate the compounds that are present in any
  sample that can be dissolved in a liquid.
• Today, compounds in trace concentrations as low
  as parts per trillion ppt may easily be
  identified.
• Separation and analysis of non-volatile or
  thermally-unstable compound

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HPLC can be applied to just about any sample,
such as
pharmaceuticals
forensic samples
environmental matrices
food
nutraceuticals
industrial chemicals
cosmetics
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• The basic components of an HPLC system include
• a solvent reservoir,
• pump,
• injector,
• analytical column,
• detector,
• recorder,
• waste reservoir.

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Isocratic SystemIn this mode, the mobile phase,
either a pure solvent or a mixture, remains the
same throughout the run.
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Gradient SystemThe mobile phase composition
changes during the separation.
High-Pressure-Gradient System
Low-Pressure-Gradient System
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Isocratic System Vs. High-Pressure-Gradient System
Gradients Can Provide Better Resolution for
Complex Sample
Solvent Strength of Mobile Phase during a Run
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HPLC System for Purification Preparative
ChromatographyCollect desired amounts of each
compound, using a fraction collector. This is
called preparative chromatography
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HPLC Instrumentation Overview
Principle Pattern
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HPLC System (Agilant 1100)
Controller
Solvent Reservoirs
Vacuum Degasser
Solvent Cabinet
Binary Pump
Recorder
Autosampler
Thermostatted Column Compartment
Detector
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Syringes
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Manual Injectors
Sample Loop
Load - Inject
Front View
Rear View
Inject
Autosampler
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HPLC - INJECTOR
Six port Rheodyne valve in which the sample
fills an external loop.
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Twin Piston Pump
The twin piston pumps with short stroke are among
the most commonly used pumps for HPLC
First pump head delivers a specific volume per
stroke. One pump is being filled while the other
is delivering the solvent.
A pressure of 40 Mpa is achieved
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Columns
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The pressure increase is inversely proportional
to the square of the particle diameter as shown
in Equation
Where, F is flow L is
column length R is column radius dp is
particle size
??? ?????? ?? 0 ?? ?? 2 ?? ?? 2
 HPLC column dimensions and the chromatographic
parameters which they influence
Van Deemter curves for various particle diameters
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• Shorter column lengths minimize all these
  variables but also reduce mechanical separation
  power.
• Column Length and Mechanical Separating Power
  Same Particle Size

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• A column of the same length but with a smaller
  particle size, will deliver more mechanical
  separation power in the same time. However, its
  backpressure will be much higher.

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Polarity Scale Polarity Scale-Mobile Phase
Polarity Scale Polarity Stationary Phase
Chromatography Mode Chromatography Mode Reversed
Phase
Chromatography Mode Chromatography Mode Normal
Phase
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Unbonded Silica Gel Particle Porous Surface
Normal Phase
C18 Bonded on Silica Non-Polar Reversed-Phase
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Detector
Variable Wavelength Detector
Refractive Index (RI) Detection
Diode Array Detector
Schematic of a Deflection Type of RI Detector
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Higher Sensitivity Detector
Mass Spectroscopy (MS)
LC-MS Spectrum of each resolved peak
Fluorescence Detector
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Welcome screen for chemstation online
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Turning on the HPLC Modules
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Preparing the HPLC System (SOP)
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Setup Pump
VWD Signal
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Pump pressure and VWD balance
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Run control gtgtgt Sample info
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Chromatogram with Isocratic Test Sample on VWD
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Chromatographic Performance Tests Resolution
(R) Resolution is de?ned as the distance between
two adjacent peak apexes, divided by the average
base width of both peaks. It is represented by
the equation Where T2 and T1 are measured in
seconds and are the peak apex retention times and
W1 and W2 are the baseline widths of the peaks,
also measured in seconds.
?? ( ?? 2 - ?? 1 ) 0.5 ( ?? 1 ?? 2 )
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Column efficiency(N) Column ef?ciency, or the
theoretical plate count, is a measure of peak
band spreading. The lower the level of band
spreading, the higher the column ef?ciency and
vice versa. There are a number of different
methods used to calculate column ef?ciency
a. Asymmetry Based ?? 41.7 ( ?? ?? ) 2 (1.25( ?? ?? )) Where x10
b. 5-Singma ??25 ( ?? ?? ) 2 Where x4.4
c. 4-Singma ??16 ( ?? ?? ) 2 Where x13.4
d. Tangent ??16 ( ?? ?? ) 2 Refer to diagram on right for W
e. 3-Singma ??9 ( ?? ?? ) 2 Where x32.4
f. Half-height ??5.54 ( ?? ?? ) 2 Where x50
g. 2-sigma (inflection) ??4 ( ?? ?? ) 2 Where x60.7
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Tailing Factor (T) Tailing factor is a measure
of the symmetry of a peak. Ideally, peaks should
be Gaussian in shape (totally symmetrical). A
peaks tailing factor is measured using the
following equation Where W0.05 peak
width at 5 height f distance from peak front
to apex point at the baseline
?? ?? 0.05 2??
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Capacity Factor (k) Capacity factor is a measure
of the retention of an analyte relative to the
column void volume, V0. It is measured using the
following equation Where,
V0 Column void volume
V1 Retention volume of peak
?? ?? 1 - ?? 0 ?? 0
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Getting the peaks perfect system suitability for
HPLC
Theoretical plates (column efficiency) More
than 2000. Resolution (Distance between two
peak) 1 to 2 min. Tailing factor Less
than 2.0. Capacity factor 2 - 8.
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METHODS ALBENDAZOLE Instrument Waters e2695 High
Performance Liquid Chromatograph Detector Waters
2998 Photodiode Array Detector Analytical
Wavelength 254 nm Column XTerra 150 x 4.6 mm,
C18 column, 3.5 µm particle size Temperature
Room temperature Isocratic Method Sample
Concentration 0.5 mg/mL Standard Concentration
(50 mg of tablet powder used to prepare 1 mg/mL
solution) Sample Solvent Acidified methanol
prepared by adding 1 mL of sulfuric acid to 99 mL
of methanol. Sample prep involves dissolving 50
mg of powder in 5 mL of acidic methanol and 25 mL
methanol in a 50 mL volumetric flask. Sonicate
for 5 minutes and bring to volume with methanol.
Mobile Phase Dissolve 0.50 g of monobasic
ammonium phosphate in 400 mL of water. Add 600 mL
of methanol, mix and filter. Bring pH to 1.7-2.0
by using a drop or two of sulfuric acid. Flow
Rate 0.75 mL/min or 1.0 mL/min Injection
Volume 15 µL Run Time 7 minutes Column
Washing After each analysis session, rinse
column with 10 column volumes of 50 water 50
methanol. Column Storage 50 methanol 50 water
after rinsing. Analytical Metrics Column
Efficiency gt2000 theoretical plates Tailing
Factor lt1.5 RSD for Replicate Injections lt0.3
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METHODS AMOXICILLIN AND AMOXICILLIN/CLAVULANATE
(OR CLAVULANIC ACID) Instrument Waters e2695
High Performance Liquid Chromatograph Detector
Waters 2998 Photodiode Array Detector Analytical
Wavelength 220 nm Column Symmetry 100 x 4.6
mm, C18 column, 5 µm particle size and
100Å Column Temperature Room temperature
Gradient Method Sample Concentration 0.5
mg/mL Amoxicillin Sample Solvent 20 mM
monobasic sodium phosphate at pH of 4.4 in 18 MO
water to make the samples. The pH is important,
as amoxicillin hydrolyzes rapidly at basic pH.
Keep standards and standard solutions
refrigerated. Use standard solutions within 3
weeks. Alternatively, freeze for storage up to 2
months. Amoxyclav Sample Solvent 100 DI
water. Clavulanate is thermally unstable. Samples
should be used within 6 hours of preparation, or
they may be held at -80C for up to 4 weeks with
lt2 degradation. Mobile Phase A 100 methanol
Mobile Phase B 20 mM monosodium phosphate
buffer, pH 4.4 in 18MO water Sample Injection
Volume 18 µL Column Washing After each
analysis session, it is important to wash out
accumulated buffer salts and degraded clavulanic
acid. Use 5 column volumes of 95 water5
methanol, 5 column volumes of 50 methanol50
water, 5 column volumes of 95 methanol5 water,
then 5 column volumes of 50 methanol50 water
again. Column Storage 60 methanol 40 water
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Gradient Description
Time (min) Mobile Phase A () Mobile Phase B () Flow (mL/min) Ramp
0.5 5 95 0.5 None
7.0-8.0 90 10 0.5 Linear
8.0-11.0 90 1 0.5 None
11.0-12.0 5 95 0.5 Linear
Analytical Metrics for Amoxicillin Column
Efficiency gt1700 theoretical plates Tailing
Factor lt2.5 RSD for Replicate Injections lt2.0
Analytical Metrics for Amoxyclav Resolution
between the amoxicillin and clavulanate peaks gt
3.5 Column Efficiency gt550 theoretical plates
Tailing Factor lt1.5 RSD for Replicate Injections
lt2.0 for both APIs
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• References
• DPAL (Distributed Pharmaceutical Analysis
  Laboratory) HPLC Methodology Manual Revised
  2015-07-14.
• Agilant 1100 series HPLC Value System Users
  Guide.
• https//www.chromacademy.com/chromatography-HPLC-C
  olumn-Dimensions.html
• 2012 Waters Corporation.
• A Troubleshooting Guide Version 1.1 Thermo
  ELECTRON CORPORATION.
• HPLC Basics courtesy of Agilent technologies,
  Inc.

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Thank you! ?