Harsh Mishra

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Presentation On HPLC After

Completion of Internship
-----By Harsh K Mishra
1 Introduction

2
Principle of HPLC

3 Columns

Content 4 4 Detector

5
System Suitability parameters

6 Method Validation Parameters

7 Project Work
Introduction

 HPLC is the form of chromatography Used to separate the components

dissolved in a solvent.

 HPLC is characterized by use of heigh pressure to push the solvent in

the stationary phase allowing separation of complex mixture with high

resolution.

 Compounds are separated by injecting the sample mixture into a

column.
PRINCIPLE

When Mixture Sample passes through passes through the column with the mobile phase as a Carrier, the
mixture is separates in its components due to adsorption on stationary Phase based on polarity, affinity &
molecular size.
High Performance Liquid Chromatography

Column

Computer

Solvent Reservoir Sampler

Pump Detector

Solvent West
Types of Elution
 ISOCRATIC - ISO => SAME - Solvent Composition Stays the Same for the Entire Run {60:40 Alcohol:

Water}

 GRADIENT - Solvent Composition Changes Throughout the Run.


1. Gradually Changed.
2. Step Chang.
Isocratic

STEP GRADIENT Gradient Curve


Columns
 Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components
of interest using various physical and chemical parameters.

 It is usually made of stainless steel to withstand high pressure caused by the pump to move the mobile phase
through the column packing other material include PEEK and glass.
TYPES OF HPLC Column

NON Polar Columns => C22, C18 , C8, Phenyl

Moderately Polar Columns => C4, C2, C18PFP, Biphenyl

Polar Columns => SILICA, Cyno, Amino,


HPLC COLUMN SELECTION
SAMPLE SOLUBILITY

Water or Polar Organic Non Polar Organic

Reverse Phase Normal Phase

 C22
 C18
 C8  Amino
 C4  Cyno
 Phenyl  silica
 Biphenyl
 Pentaflourophenyl
Normal Phase HPLC Columns :
 This type of columns has more polar stationary phase than the mobile phase . silica that is polar material is used
as Packing Material But water is more polar than the silica , therefore , water is not used and methylene
chloride , hexane and chloroform or a mixture of these with diethyl ether is used as mobile phase .

 Separation of the sample components occurs on the basis of the polarity of the sample components . Sample
components having more polarity interact more with polar stationary phase resulting in separation from the less
polar component.
Reverse Phase HPLC Columns
 Reverse phase columns as its name states , it is reverse of the normal phase columns . It has a non -
polar stationary phase and polar mobile phase . Bonded hydrocarbons like C8 and C18 and other
non polar hydrocarbons are used as stationary phase in reverse phase columns while aqueous
organic solution like used as mobile phase.

 Separation of sample components in reverse phase column also occurs on the basis on the polarity
of the sample components but it happens just opposite of the normal phase HPLC columns ,
therefore , this type of chromatography is known as Reverse Phase Chromatography .
Column Description Application
C18 Optimized for Maximum efficiency, superior peak ship and A C18 Phase for most HPLC
resolution application. Available in Range of
particle size, from LC/MS and
preparative scale separation.

C8 Increased bonding density compare to C18, Optimized for Recommended starting point for
Maximum efficiency, superior peak Shap and resolution method development. Also suited
to high aqueous condition and for
Rapid analysis application.

C4 Combine lower hydrophobicity with excellence Used for rapid analysis


chromatography performance. Improved hydrolytic optimization, when less retention
stability compared to conventional C4 phase than C8 or C18 is required. Also
suitable for analysis of small
proteins.
Column Description Application
CN Suitable for use in both normal- and Use to increase retention of polar
reversed modes . Greatly improved compounds . Ideal for gradients and
performance , stability and rapid screening applications due to
reproducibility compared to fast equilibration capabilities.
conventional CN phases

Ph Hydrophobicity between C4 and C8 Offers alternative selectivity for


phases , with increased polar aromatic , amine or polar
selectivity . Improved compounds
performance , stability and
reproducibility compared to
conventional phenyl phases

AQ unique C18 bonded phase with Recommended for applications


integral polar functionality . where 100 % aqueous mobile
Resistant to phase collapse even phases are required . Ideal for fast
with 100 % aqueous mobile phase . gradients due to rapid re
equilibration properties .
Detectors
Different compound needs different detectors

I. UV Detector
II. PDA Detector
III. Fluorescence detector
IV. RI detector
V. Evaporative light scattering Detector
UV Detector

According To beer Lambert law

A = ɛ x l x c.
A= Absorption
ɛ= Molar absorptivity
C= Concentration
Molar Absorptivity Value Of Some Compounds

A = ɛ x l x c.
Photo Diode Array Detector

 It can analyse sample at different


wavelength simultaneously .

 They are Mostly Used for


Method Development

 PDA Detector are used to check


peak purity.

 Relatively Robust to fluctuation


in flow rate and temperature.
Refractive Index Detector

 Snell's Law which states that when Light Passes From Rear
Medium To denser Medium It Bends Toward Normal.

 These Indicators are called Universal indicator

 Use for Detection of lipids, Alcohols, natural compounds


like carbohydrates.
Fluorescence Detector

 These Detectors are extremely sensitive detector,

Making Ideal for Trace Analysis and Complex

Sample Matrix.

 Can Detect Sample In ng/ml and pg/ml.

 It is used for detection of compounds Like sugar,

Lipids, carbohydrates.
Evaporative Light Scattering Detector
 Evaporative Light Scattering Detector also known
as Universal Detector.

 Compound Having ɛ <100 are not Detected by

PDA detector hence ELS detector are used.

Drawbacks

 Cannot Used for Volatile Samples.


 Mobile Phase having Volatile nature cannot be

used used in this analysis.


System Suitability Criteria
 Retention time
 Retention factor
 Repeatability (% RSD)
 Resolution
 Tailing factor
 Theoretical plates
 Signal-to-Noise ratio
Retention Time(TR)

In liquid chromatography and gas chromatography, the retention time, TR1 TR2, is
defined as the time elapsed between the injection of the sample and the appearance of
the maximum peak response of the elution sample Zone.
Resolution
 The resolution is the separation of two components in a mixture.

Rs= 2(tR2-tR1)
(W1+W2)
 For good Gaussian peak Rs should be greater than 1.5
Retention factor
 The retention factor is equal to the ratio of retention time of the analyte on the column to the
retention time of a non-retained compound.
 The non-retained compound has no affinity for the stationary phase and elutes with the solvent front
at a time t0, which is also known as the ‘hold-up time’ or ‘dead time’.
Tailing factor

For related substance test or assay, the tailing of standard peak


used for quantification should be 0.8 to 1.5

w 0.05
AS = ----------
2f

w 0.05
= width of the peak at one- twentieth of

the peak height (5% height) ,


f = Distance from the peak maximum to
the leading edge of the peak, the distance being measured
at a point 5% of the peak height from the base line
Repeatability RSD
 Unless otherwise specified in the individual monograph,

 For 5 replicate injections, RSD < 2.0%

 For 6 replicate injections, RSD > 2.0%


Number of (N) theoretical plats
 Is a measure of column efficiency

t = is the retention time of the


substance

w = is the width of the peak at its base, obtained by extrapolating the relatively straight
sides of the peak to the baseline.

 According to USP Number of Theoretical Plate should be 15163 for good Column efficiency.
Signal to Noise Ratio
A useful system suitability parameter
2H
S/N = --------
hn
H =Height of peak measured from the
maximum of the peak to the extrapolated base line of the signal
observed over a distance equal to at least 5 times the width at half
height.

h or(hn) =Range of the noise in a chromatogram obtained after


injection of a blank, observed over a distance equal to at least 5
times the width at half height.
Method validation Parameters

Accuracy

Accuracy of the measurement is define as closeness of the measured value to the


true value.

Method

Accuracy should be assessed using a minimum of 3 concentration levels covering


the specified range.

50%
75%
100%
125%
Precision
 The precision of an analytical procedure expresses the
closeness of agreement between a series of
measurements obtained from multiple sampling of the
same homogeneous sample under the prescribed
conditions. Precision may be considered at three levels:

Method
 Repeatability
 Intermediate Precision
 Reproducibility

 6 to 15 Measurement are taken for single Sample at


Each Concentration and RSD is calculated
 For assay Method RSD <2%
Linearity
The linearity of an analytical procedure is its ability (within a given range) to obtain
test results which are directly proportional to the concentration (amount) of analyte in
the sample.

Method
 Sample Preparation Of different
Concentration
 5,10,15,20,25ppm
 Are injected and correlation
coefficient are calculated
 It should Not be less than 0.999
LOD
 The detection limit of an individual analytical procedure is the lowest amount
of analyte in a sample which can be detected but not necessarily quantitated as
an exact value.
LOD = 3.3 * σ/s

LOQ

The quantitation limit of an individual analytical procedure is the lowest amount of


analyte in a sample which can be quantitatively determined with suitable precision and
accuracy.

LOQ = 10 * σ/s
Robustness
The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small, but deliberate variations in method parameters and provides an
indication of its reliability during normal usage.

Method
 Temperature

 Flow Rate

 Mobile phase composition


PROJECT
Method development and validation for Assay of Raw Material In
Drug “X” by HPLC
HPLC
Instrument I. Agilent
II. Waters

Software  Empower
Mobile Phase  MP A – 0.1%Perchloric Acid
MP B – Acetonitrile
Diluent  Methanol & water (90:10)
Temperature  250 c
Wavelength  260nm
Flow Rate  1.5 ml/min
Column  C-8 250*4.6mm,
Plan Of Work
Step 1: Physical
Characterization and Step 3: Method Validation
Properties

Step 2: Analytical Method System suitability


 Appearance • Specificity

Development
Color Step 4:
 Solubility • Precision
To undertake solubility studies for Compilation
 Melting point • Linearity Of data
analyte. • LOQ (Limit of Quantification)  Result
• Selection Of suitable Mobile Phase • Accuracy  Discussion
• Selection of suitable solvents. • Recovery  conclusion.
• Column selection • Stability study
• Develop initial conditions for HPLC
method.
• Optimization of the HPLC method.
Thankyou

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