The document discusses the qualification of high performance thin layer chromatography (HPTLC). It describes the four types of qualification: design qualification, installation qualification, operation qualification, and performance qualification. Design qualification verifies specifications and review methods. Installation qualification documents compliance at installation. Operation qualification documents consistent performance within operating ranges. Performance qualification ascertains the instrument is suitable for specific analytical tasks. The document then provides examples of tests to check HPTLC performance, including linearity of spotting, reproducibility of spotting, and detection capacity.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
QUALIFICATION OF HPTLC
A Seminar as a part of curricular requirement
for I year M. Pharm II Semester
Presented by
G. Sateesh Chandra
(Reg. No. 20L81S0711)
Dept. of Pharmaceutical Analysis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Principle
• Salient features of HPTLC
• Steps involved in HPTLC development
• Instrumentation
• Applications of HPTLC
• Qualification
• References
Contents
1. Design Qualification (DQ)
2. Installation Qualification (IQ)
3. Operation Qualification (OQ)
4. Performance Qualification (PQ)

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• HPTLC have similar approach and employ the same physical
principles of TLC (adsorption chromatography) i.e. the principle of
separation is adsorption.
• The mobile phase solvent flows through because of capillary action.
The components move according to their affinities towards the
adsorbent.
• Thus the components are separated on a chromatographic plate.
Principle
The component with more affinity towards the stationary phase
travels slower.
The component with lesser affinity towards the stationary phase
travels faster.

4. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
Salient features of HPTLC
• It is simple to learn and operate.
• Accuracy and precision of quantification is high.
• Samples rarely require cleanup.
• Low maintenance cost.

5. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• Selection of chromatographic layer.
• Layer pre-washing of precoated plates.
• Layer Pre-conditioning/Activation of precoated plates.
• Selection and Optimization of mobile phase.
• Sample and standard preparation
• Application of sample and standard.
• Chromatographic development.
• Detection of spots.
• Scanning and documentation of chromo plate.
Steps involved in HPTLC
development

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
• Sample applicator
• Developing chamber
• Derivatization device
• Immersion device
• Plate heater
• Scanning densitometer
• Other accessories like: (i) Plate coater
(ii) Drying rack
(iii) Plate cutter
Instrumentation

7. RIPER
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NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
• Herbal fingerprinting.
• Herbal Analysis – Quantification.
• Pharmaceutical Science.
• Determination of purity of sample and Identification of compounds.
• Identification of adulterants.
• Forensic science.
• Determination of mercury in water.
• Analysis of environmental pollution levels.
• Determination of ß-blockers like Metaprolol, Alprenolol, Atenolol.
Applications of HPTLC

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
• Qualification is a part of validation. It is action of proving and
documenting the equipment which are properly installed.
• Steps involved in qualification are:
Qualification
Design Qualification
Installation Qualification
Operation Qualification
Performance Qualification

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• This qualification verifies that the rigorous specifications and design
review methods defined in the Quality Management System of the
manufacturer have been followed.
• Certified Quality Management System ascertains planned testing
procedures, error reporting and controlled updating of documents.
• Compliance is documented
e.g. by the "Declaration of System Validation" and
"Declaration of Conformity" supplied with specific products.
Design Qualification (DQ)

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• The Design Qualification is sometimes used in a different meaning.
One common misunderstanding is to use DQ for "suitability of the
laboratory equipment".
• To make sure the laboratory is equipped with the necessary
supporting equipment etc.
Cont…

11. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
• This qualification is performed at the site and time of installation.
• It documents that all key aspects of the installation comply with the
manufacturer’s specifications, codes, safety and design parameters.
• In order to qualify for an IQ Certificate, this procedure is to be
performed by a Product Specialist.
Installation Qualification (IQ)

12. RIPER
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NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
• This qualification is performed subsequent to installation and is
repeated at certain intervals recommended by the manufacturer or
defined by the customer.
• It documents that all modules of the equipment perform consistently
throughout the specified operating ranges.
• The initial OQ is performed by the person responsible for the IQ at
installation.
• In order to qualify for an OQ Certificate, this procedure is to be
performed by a Product Specialist, approved by manufacturer.
Operation Qualification (OQ)

13. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
• Repetitive OQ’s can be performed by a system user well aquatinted
with the system, following guidelines issued by manufacturer.
• On request of the customer, such OQ’s can be performed by a
Product Specialist or Service Engineer, against a fee or within a
service contract.
Cont……

14. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• PQ is performed to ascertain that the instrument (system) is suitable
to perform a specific analytical task as part of the manufacturing
process.
• PQ is an on-going task with the customers samples and procedures
including preventive maintenance and regular tests, such as system
suitability and quality control analyses with creation of QC-charts.
• For computer systems it also includes regular data backup, virus
checks and change control procedures.
• PQ can thus only be performed by the user himself who also has to
create the SOP’s based on the analytical task, procedure, the different
instrument manuals and the customer's QC requirements.
Performance Qualification (PQ)

15. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
Check the performance of HPTLC in terms of
1. Linearity of spotting
2. Reproducibility of Spotting
3. Detection capacity
Cont…

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
1. Linearity of spotting
• Apply 2 µl, 4µl, 6µl, 8µl and 10 µl of solution on HPTLC plate with
spotter.
• Allow the plate to run in mobile phase.
• Dry the plate with drier.
• Scan the plate with scanner.
• Check the linearity and correlation coefficient between the spots.
• How to check linearity and correlation coefficient b/w the spots??
Correlation Coefficient:
• A number that gives you a good idea about how closely one
variable(spot) is related to another variable.

17. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
• Acceptance limit
• Correlation coefficient = Not less than 0.9900
Check the linearity of spots

18. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
2. Reproducibility of spotting
• Apply 10µl solution on HPTLC plate for six times in sequence.
• Allow the plate to run in mobile phase.
• Dry the plate with drier.
• Scan the plate with scanner.
• Calculate the Relative Standard Deviation(RSD) for six tracks.
Acceptance limit
• Documentation of RSD, RSD Limit : NMT 3.0%

19. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
3. Detection capacity
• Requirements
i. Alumina glass plate
ii. Sodium salicylate
iii. 96% v/v alcohol
• Preparation of stock solution
Stock solution-1:
Weigh 500mg of Sodium salicylate and transfer it into 250ml
volumetric flask dissolve and dilute with 96% v/v alcohol.
Stock solution-2:
Weigh 100mg of Sodium salicylate and transfer it into 250ml
volumetric flask dissolve and dilute with 96% v/v alcohol.

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
• Procedure
Spot 5 micro liter of each solution observe at 254nm and 366nm.
Acceptance:
1. The spot shall be comparable intensity wise.
2. Spot due to stock solution-2 shall be visible at 254nm.
3. Spot due to stock solution-1 shall be visible at 366nm.

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
• D. Gowrishankar, K. Abbulu, ett al. Validation and calibration of
analytical instrument. 2010: 2(2).
• B. K. Sharma, Instrumental chemical analysis, 2012(1).
• Herman lam, ett al. Analytical method validation and instrument
performance verification,153-186.
References

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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22