Harsh Mishra
Harsh Mishra
Harsh Mishra
Completion of Internship
-----By Harsh K Mishra
1 Introduction
2
Principle of HPLC
3 Columns
Content 4 4 Detector
5
System Suitability parameters
7 Project Work
Introduction
dissolved in a solvent.
resolution.
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
Pump Detector
Solvent West
Types of Elution
ISOCRATIC - ISO => SAME - Solvent Composition Stays the Same for the Entire Run {60:40 Alcohol:
Water}
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
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.
I. UV Detector
II. PDA Detector
III. Fluorescence detector
IV. RI detector
V. Evaporative light scattering Detector
UV Detector
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
Snell's Law which states that when Light Passes From Rear
Medium To denser Medium It Bends Toward Normal.
Sample Matrix.
Lipids, carbohydrates.
Evaporative Light Scattering Detector
Evaporative Light Scattering Detector also known
as Universal Detector.
Drawbacks
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
w 0.05
AS = ----------
2f
w 0.05
= width of the peak at one- twentieth of
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.
Accuracy
Method
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
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
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
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