Presentation of HPLC

CRCL Group A Officer’s Training at IICT
Welcome to All
Distinguished Faculty Members of IICT
& Officers of Revenue Laboratories.
05/04/2019 1IICT Training Presentation-2 Group-III

Don’t worry Singh Sir, I
have some knowledge
about HPLC, we will
discuss and our team
work together to get a
better solution
Mr. Suresh, I have one
sample and I need a
quantitative separation of
it, have you any idea about
that?

Topic: High Performance Liquid
Chromatography(HPLC)
Group-III
Team Members:
1. T.R Suresh
2. A.J Aleyamma
3. Ajay Kumar Singh
4. H S Bhandarkar
5. K Thambidurai

Topic to be Covered
1. Introduction
2. Basics of HPLC
3. Types of Liquid Chromatography
4. Instrumentation
5. Application

Introduction
HPLC is a form of liquid
chromatography used to separate compounds
that are dissolved in solution.
HPLC instruments consist of a reservoir of
mobile phase, a pump, an injector, a separation
column, detector and a recorder or output
device.

Basic Principles of HPLC
Stationary Phase - the phase which remains fixed in the
column, e.g. C18, Silica
Mobile Phase - carries the solvent along with the sample
through the stationary phase as it moves through the column.
Compounds are separated based upon their differential
migration / partition behaviour between the stationary
and mobile phases.

Types of Liquid Chromatography
 Partition chromatography
 Adsorption, or liquid-solid chromatography
 Ion exchange chromatography
 Size exclusion, or gel, chromatography

In Partition chromatography, the molecules are separated in between two phases based
ion the partition coefficient of the analyte.
The partition chromatography is the basic principle involved in many separation
techniques like high performance liquid chromatography and gas chromatography.

 Adsorption, or liquid-solid
 Here separation is based on the adsorption/desorption
of the analyte onto a polar surface(silica). The solid surface
is called adsorbent which is generally porous in nature with a
high surface area to adsorb substances on its surface
by intermolecular forces .
 Some commonly used adsorbents are Silica gel H, silica
gel G, silica gel N, silica gel S, hydrated gel silica, cellulose
microcrystalline, alumina, modified silica gel, etc.

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0IICT Training Presentation-2 Group-III
 Ion Exchange chromatography
In this type of Chromatography the sample
components are separated based upon ionic
interaction and exchanging of ions between the
charged groups of molecules of analyte and
charged groups in the stationery phase opposite
opposite to that of analyte molecules. Polar
mobile liquid, usually water containing salts or
small amounts of alcohols, and a stationary phase
containing either acidic or basic fixed sites.

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1IICT Training Presentation-2 Group-III
Size Exclusion (OR) Gel chromatography (Gel Permeable )
Seperation based on analyte’s molecular size and sieving
action of the column packing. In size exclusion the column is
consisted of substances which have controlled pore sizes and is
able to be filtered according to its molecular size. Small
molecules penetrate into the pores within the packing while
larger molecules only partially penetrate the pores. The large
molecules elute first before the smaller molecules.

05/04/2019 IICT Training Presentation-2 Group-III
Instrumentation
Schematic Diagram of working principle:

Instrumentation
Mobile Phases
Pump / Mixing Chamber
Injector port
Column Chamber
Recorder / Control System

Instrumentation
Components of HPLC:
 Solvent
 Solvent Delivery System (Pump)
 Injector
 Sampler
 Column
 Detectors (Diode Array)
 Fraction Collector
 Recorder (Data Collection)

Instrumentation
Components of HPLC:
 Solvent (Mobile Phase):
 Mobile phase is usually a mixture of one or more solvents and it should be High,
Purity, Low Viscosity and Low Boiling Points.
 The Common Solvents used are N-Hexane, Toluene, Methylene Chloride, THF,
ACN, Methanol and Water
 Binary system contains Two Solvents, and Quaternary System Contains Four
Solvents

Instrumentation
Components of HPLC:
 Pump:
 Pump is a important device used to mix the solvents, to maintain the
required flow rate of Mobile phase in the system.

Instrumentation
Components of HPLC:
 Injector and Sampler: [Autosampler, Sample Manager]
 Injector – It is a simple device to introduce the sample
volume of a sample solution into the flowing mobile
phase stream. The injector can be a simple manual
device, or a sophisticated autosampler that can be
programmed for unattended injections of many samples
from an array of individual vials.

Instrumentation
Components of HPLC:
 Column: Types of Columns
–Microbore
•Diameter 1-2 mm; Length 7-30 cm; sample size 0.01
mg; flow rate 0.1 mL/min
–Std. analytical
•Diameter 3-5 mm; Length 7-30 cm; sample size 0.1 mg;
flow rate 1 mL/min; Particle Size 3-10 μm
–Preparative
•Diameter 5-20 mm; Length 25-50 cm; sample size 10
mg; flow rate 10 mL/min ; Particle size >10 μm

Instrumentation
Components of HPLC:
 Column: Column Packing Material
•Support Type
–Silica (or alumina) or polymer (cross-linked polystyrene)
•Bonded groups
–C18, C8, C4, amino, cyano, phenyl
–Diethylaminoethyl (DEAE), sulfonate, quaternary ammonium
•Particle size (dp): 3-, 5-, 7,-10-or 20 μm
–Efficiency is inversely proportional to dp
–Column pressure is inversely proportional to (dp)2
•Pore size (dpore): 60-300A
–Wide pore materials (300 A) are used for biomolecules or polymers
•Surface area: 90-400 m2/g
–High surface area maximizes solute interaction with bonded groups

Instrumentation
Components of HPLC:
Column Length (L)
•Column efficiency (n) is proportional to Length
•Common column length is 10-25 cm
•Column can be connected together to produce a longer
column with higher efficiency
•Column back pressure is proportional to L
•Fast LC column are short column (3-10 cm) packed with
small-particles (dp= 3 μm)

Instrumentation
Components of HPLC:
Column Diameter (d)
 Sample capacity is proportional to (d)2
 Flow rate is proportional to (d)2
 Detection limits is inversely proportional (d)2
Commonly used column with following diametrs:
Dia Sample Wt. Flow Rate
For Semi-preparative 8-20 mm 10-50mg 5-30 ml/min
Standard Analytical 4.6 mm 1 mg 1 ml/min
Narrow bore 2.0 mm 0.2 mg 0.2 ml/min
Mirco bore 1.0 mm 0.05 mg 0.05 ml/min

Instrumentation
Components of HPLC:
Detector
 A device that indicates the composition of
the eluent by measuring physical or
chemical properties [e.g., UV/visible light
absorbance, differential refractive index,
fluorescence, or conductivity].

Instrumentation
Components of HPLC:
Types of Detector
 UV-Visible Detector
Fluorescence Detector
Photo-Diode Array (PDA)
Evaporative Light Scattering Detector (ELSD)
Refractive-Index (RI)
Mass-Spectrometric (MS)

Instrumentation
Components of HPLC:
Types of Detector
 UV-Visible Detector - For Unsaturated Organic & Carbonyl Compounds
Fluorescence Detector – It is one of the most sensitive detector and
often used for trace analysis.
Photo-Diode Array (PDA) - The diode arrays work in parallel
simultaneously monitoring all the
wavelengths
Evaporative Light Scattering Detector (ELSD)
- Universal detection of non-volatile compounds
Refractive-Index (RI)
- this detector is extremely useful for detecting those compounds that
are nonionic, do not adsorb in the UV, and do not fluoresce.
Mass-Spectrometric (MS)

Instrumentation
Fraction Collector:
Fraction collector is used to collect the different fraction of the compounds based
on their retention time in semi preparative scale

Application of HPLC

Mobile phase: Dissolve 1.0 g of sodium heptane sulfonate monohydrate R
in 420 mL of water, adjust to pH 3.2 with phosphoric acid
(4.9 g/L H3PO4) (about 5 mL) and add 180mLof acetonitrile.
Stationary phase: Octylsilyl silica gel for chromatography R(5 μm). C-18 Column
Flow Rate: 1.5 ml / Minute
Detection: Spectrophotometer at 280nm
Reference solution: Dissolve 25.0 mg (M1) of Morphine Standard in the mobile
phase and dilute to 25.0mL(V1) with the mobile phase.
Dilute 10.0 mL(V2) of this solution to 100.0 mL (V3) with the
mobile phase.

Sample Solution Preparation:
Suspend 1.00 g(M2)of the substance to be examined, cut into thin slices,
in 50 mL of ethanol (50 per cent V/V) ,mix with the aid of ultrasonic bath
for 1 hours, allow to cool and dilute to 100.0 mL(V4) with the same
solvent. Allow to stand.
To 10.0 mL(V5)of the supernatant liquid add 5 mL of ammonium chloride
buffer solution pH 9.5 R, dilute to 25.0 mL (V6) with water and mix.
Transfer 20.0 mL (V7) of this solution to a chromatography column about
0.15 m long and about 30 mm in internal diameter containing 15 g of
kieselguhr for chromatography. Allow to stand for 15 min. Elute with 2
quantities, each of 40 mL, of a mixture of 15 volumes of 2-propanol R and
85 volumes of methylene chloride R. Evaporate the elute to dryness in
vacuum at 40 °C using rotary evoporator. Transfer the residue to a
volumetric flask with the aid of the mobile phase and dilute to 25.0 mL
(V8) with the mobile phase. Inject this sample in HPLC.

29
HPLC Chromatogram for Standard Morphine
Morphine

IICT Training Presentation-2 Group-III05/04/2019 30
HPLC Chromatogram for Raw Opium Sample
Morphine

IICT Training Presentation-2 Group-III
05/04/2019 31
Calculation

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Typical Analysis of Different Raw Opium Samples

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Presentation of HPLC

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