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HPLC and HPTLC standardization of crude drug

HPLC and HPTLC standardization of crude drug Dr. Mohan G. Kalaskar R. C. Patel Institute of

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

 Principle – – St phase – liquid – Mobile phase – liquid  partition coefficient
  • 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)

H PLC system

H PLC system  Solvent Reservoir  Degasser  Solvent Delivery System (Pump)  Injector 
  • Solvent Reservoir

  • Degasser

  • Solvent Delivery System

(Pump)

  • Injector

  • Column &oven

  • Detectors

  • Recorder (Data Collection)

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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
Kalmegh
Qualitative determination RT- 3 min Uv – 254 nm
Qualitative determination
RT- 3 min
Uv – 254 nm
Quantitative determination  Standard preparation – – Prepare the gradual increased dilutions of andrographalide of known
Quantitative determination
Standard preparation –
Prepare the gradual increased dilutions of andrographalide of known conc (20-100
µg/ml) in methanol
Sample preparation
Known conc of extract (100 µg/ml)
Procedure
Inject 20 µl of different conc. of standards followed by sample
plot calibration graph of standard (AUC Vs Conc)
Standard RT- 3 min Uv – 254 nm
Standard
RT- 3 min
Uv – 254 nm
Test extract RT- 3 min Uv – 254 nm
Test extract
RT- 3 min
Uv – 254 nm
 – Where • Y= AUC of unknown • X= conc. of unknown
– Where
• Y= AUC of unknown
• X= conc. of unknown

Regression equation – Y=MX + C X= (Y-C)/M

HPLTC

HPTLC

-

Less affinity Moderate affinity More affinity
Less affinity
Moderate affinity
More affinity

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

Difference between HPTLC & TLC  HPTLC TLC  Particle size 4-8µm 5-20µm  Sorbent layer
Difference between HPTLC & TLC
HPTLC
TLC
Particle size
4-8µm
5-20µm
Sorbent layer
thickness
100µm
250µm
Efficiency
High
Less
Separations
3-5cm
10-15cm
Analysis time
Faster
Slower
Development
chamber
Less amount of
mobile phase
Sample spotting
Auto sampler
Scanning.
Densitometer
More amount
of mob. phase
Manual spotting
short or long Uv
3/13/20
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DI FFERENCE BETWEEN H PTLC & HPLC HPTLC HPLC Simultaneous processing of sample & standard simultaneous
DI FFERENCE BETWEEN
H PTLC & HPLC
HPTLC
HPLC
Simultaneous processing
of sample & standard
simultaneous process
of sample & std. not
under same condition
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
s
ample
of differernt volume can
sample of same vol only applied
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can applied
Activation of plates
Activation of plates
K almegh

K almegh

Standard
Standard