HPLC and HPTLC standardization

of crude drug
Dr. Mohan G. Kalaskar
R. C. Patel Institute of Pharmaceutical Education and Research,
Shirpur, MS, India

HPLC
originally referred to:
High Pressure Liquid Chromatography
high pressure to be able to use small particle size to allow proper
separation at reasonable flow rates
Laterly referred to:
High Performance Liquid Chromatography
high performance due to its reproducibility

Compounds are separated by injecting a sample mixture onto

the column.

The different component in the mixture pass through the column

at different rates due to differences in their partition behavior
between the mobile phase and the stationary phase.

Thus separated

Principle
St phase liquid
Mobile phase liquid

partition coefficient
is the ratio of concentrations of a compound in a
mixture of two immiscible phases

Partition (liquid-liquid)
Reverse-phase
Non-polar stationary phase

Normal-phase
Polar stationary phase

Components Of A Liquid Chromatograph System

Mobile Phase / Solvent Reservoir

Degasser

Solvent Delivery System (Pump)

Injector

Precolumn

Column

Temperature Control

Detectors

Recorder (Data Collection)

HPLC system

Solvent Reservoir
Degasser
Solvent Delivery System
(Pump)

Injector

Column &oven

Detectors
Recorder (Data Collection)

Advantages of HPLC

Higher resolution and speed of analysis

HPLC columns can be reused without repacking or

regeneration

Greater reproducibility due to close control of the

parameters affecting the efficiency of separation

Easy automation of instrument operation and data

analysis

Adaptability to large-scale,

preparative procedures

Kalmegh

Qualitative determination
RT- 3 min
U v 254 n m

Quantitative determination

Standard preparation
Prepare the gradual increased dilutions of andrographalide of known conc (20-100
g/ml) in methanol

Sample preparation
Known conc of extract (100 g/ml)

Procedure
Inject 20 l of different conc. of standards followed by sample
plot calibration graph of standard (AUC Vs Conc)

Standard
RT- 3 min
U v 254 n m

Test extract
RT- 3 min
U v 254 n m

Regression equation Y=MX + C

X= (Y-C)/M
Where
Y= AUC of unknown
X= conc. of unknown

HPLTC

HPTLC -

High Performance Thin Layer Chromatography.

Sophisticated & automated form of

TLC.

Principle
Separation may result due to
adsorption or partition or by both
phenomenon depending upon the
nature of adsorbents used on plates
and solvents system used for
development.
Adsorption- based on relative
affinity toward stationary phase

Less affinity

Moderate affinity

More affinity

Difference between HPTLC & TLC

Particle size
Sorbent layer
thickness
Efficiency
Separations
Analysis time
Development
chamber
Sample spotting
Scanning.

3/13/2015

HPTLC
4-8m
100m
High
3-5cm
Faster
Less amount of
mobile phase
Auto sampler
Densitometer

TLC
5-20m
250m
Less
10-15cm
Slower
More amount
of mob. phase
Manual spotting
short or long Uv

15

DIFFERENCE BETWEEN
HPTLC & HPLC
HPTLC
Simultaneous processing
of sample & standard
under same condition

HPLC
simultaneous process
of sample & std. not
possible

Extreme flexibility for

various steps

Limited flexibility

Technically, it is simple
to learn & operate

Skilled & well-trained

personnel are needed

sample preparation is simple

sample preparation is critical

sample of differernt volume can

can applied

sample of same vol only applied

16

Activation of plates

Kalmegh

Standard