validation HPLC and HPTLC

1. PRESENTED BY:-
DEVA P. PUNDKAR
M-PHARM QUALITY
ASSURANCE
ROLL NO 13
TOPIC:- QUALIFICATION OF
ANALYTICAL INSTRUMENTS HPLC
AND HPTLC
SANDIP INSTITUTE OF
PHARMACUTICAL SCIENCE

2. contents

3. Introduction

4. Introduction
QUALIFICATION:
Qualification is part of validation; Action of providing and
documenting that equipment or ancillary systems are properly
installed, work correctly and actually lead to the expected results.
The entire qualification consists of four parts:
1.Design qualification (DQ)
2.Installation qualification (IQ)
3.Operational qualification (OQ)
4.Performance qualification(PQ)

5. HPLC

6. HIGH PERFORMANCE LIQUID CHORMATOGRAPHY
(HPLC):
• HPLC is a form of column chromatography that pumps at high
pressure a sample (analyte) dissolved in a solvent (mobile phase)
through a column with an immobilized chromatographic packing
material.
• As the sample passes through the column, analytes having the
strongest interactions with the stationary phase exit the column
slowest, means they exhibit the longest retention time.
• Retention time of analyte depends on sample, solvent, nature of
stationery phase.

8. DESIGN
QUALIFICATION

9. DESIGN QUALIFICATION
• It describe the user requirements and defines the
functional and operational specifications of the
instrument.
• DQ should ensure that instrument to be purchased
have the necessary functions and performance that
will enable for suitable intended application.

10. Design element Example
Intended use Analysis of drug components and impurities.
User requirements specification for the HPLC
analysis
• Up to 100 samples/day Limit of
quantitation:0.1%Automated over night
analysis.
• Automated confirmation of peak identity and
purity with diode-array detection
• Automated compound quantitation and
printing of report.
FUNCTIONAL SPECIFICATION:
• Pump
• Detector
• Auto sampler
• Colum compartment
• computer
• Binary or higher gradient
• UV/VIS Diode array, 190-900nm 100 samples, 0.5ul
to 5ml sample
• volume15 to 60°c controlled.
• System control, data acquisition for signals and
spectra, peak integration and quantitation

11. Design element Example
Spectral evaluation for peak purity and
compound confirmation. Electronically save all
chromatograms generated by the system
Operational specification • Detector: base line noise:<5 x 10-5AU
• Sampler: precision inj. Volume: <0.5% RSD.
Sample carry over:<0.5%
• Pump: precision of retain time: <0.5% RSD
AU= ABSORBANCE UNIT
RSD= RELATIVE STANDRD DEVIATION
• = • Operation manual on paper
• Computer based tutorial
• The vendor must provide procedures and
services for IQ and OQ

12. Design element Example
Maintenance •Vendor must deliver maintenance
procedure and recommended schedule
•Instrument must include early
maintenance feedback for timely
exchange of most important maintenance
parts.
Maintenance procedure must be supplied
on multimedia CD ROM
Training •Vendor must provide familiarization and
training

13. Installation
qualification

14. Installation qualification establishes
• That the instrument is received as design and specified.
• Installed in the selected environment and that should be suitable
for operation of instrument.
Installation qualification

15. Before
installation
Obtain manufacturing
recommendation for
installation
Check the site for fulfilment for
utilities such as electricity,
temperature etc
Allow sufficient space for equipment, SOPs
operatio manual and software

16. During
installation
• Compare equipment as received
with purchase order.
• Check documentation for
completeness
• Check equipment for any damage
• Install hardware.
• Check all modules power up
• Run test sample and
chromatogram with reference
• Prepare an installation report

17. Operation
Qualification

18. Operation Qualification
• It is the process of demonstrating that an instrument will
function according to its operational specification in the
selected environment.
• It verifies that the HPLC system compiles with key function
and operational requirements as specified in the design
qualification.
• Before performing all other test first perform leak test if, it is
failed then most of the remaining test will get failed

19. Parameter Procedure User limit
Leak testing Flow test by volume or
weight/time
+- 5
Baseline drift ASTM (American society
for testing material)
method E19.09, 20 min.
<2 x 10-3 AU
Baseline noise ASTM method E19.09,
20 min x 1
<5 x 10-5 AU
Precision of
injection volume
6 x injection of caffeine
standard, RSD of peak
areas
0.3 % RSD
Precision of flow
rate
6 x injection of caffeine
standard, RSD of
retention times
0.5% RSD
TEST PARAMETERS AND ACCEPTANCE CRITERIA:

20. Parameter Procedure User limit
Temperature precision Monitoring
temperature over 20
mins
+-0.25 c
Auto sampler carry
over
Injection of sample
after large
concentration
<0.5%
Mobile phase
composition accuracy
Step gradient from 4 to
7% B, step heights
relative to 100% with
acetone trace
+-1%

21. Performance
qualification

22. Performance qualification
Category Objective Exactions
System check Display the result of the system check,
including the use of defected parts, UV-
VIS wavelength accuracy and other
validation items, to verify that the
selection analysis are satisfied
Before analysis daily
Performance check • Flow rate accuracy
• solvent delivery stability
• Pressure limits
• Gradient concentration
• Column oven temperature accuracy
and control
• Leak test of pips
• UV-VIS detector wavelength accuracy
/D2 lamp

23. Category Objective Exactions
Performance check • Absorbance accuracy using
internal filter
• Absorbance accuracy using
external filter
• Bassline drift noise check
• Auto sample injection volume
accuracy
• Sample cooler temperature
accuracy
• Auto sampler
• Detector linearity
Periodically or when
instrument problem arise
Auto validation • Performance items which can be
performed automatically are
selected either isocratic or
gradient delivery mode is selected

24. Category Objective Exactions
Supplemental check Injection volume
repeatability check using
resistor tubulin instead of a
column
Periodically

25. HPTLC

26. HPTLC
STEPS INVOLVED IN HPTLC DEVELOPMENT
• 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.

28. INSTRUMENTATION
• Sample applicator
• Developing chamber
• Derivatization device
• Immersion device
• Plate heater
• Scanning densitometer
• Other accessories like:
• (i) Plate coater
• (ii) Drying rack
• (iii) Plate cutter.

29. SAMPLE APPLICATOR
• The samples are applied onto the layer as spots or bands.
• Usual concentration of applied samples 0.1 to 1 µg/μl for
qualitative Analysis and quantity may vary in quantitation based on
UV absorption to 5 μl for spot and 10 μL for band application.
• Three types of sample applicators are available:
1.Manual
2.Semiautomatic
3. automatic

30. DEVELOPING CHAMBER
• The "classical" flat-bottomed chamber is
available in many sizes from various
manufacturers.
• It has two types:-
1.Twin Trough Chambers (TTC)
2. Automatic Developing Chamber (ADC)

31. 1. Twin Trough Chambers (TTC)
Twin trough chambers (TTC) (CAMAG) are among the most
widely used chambers.
• They are available in sizes10 *10 cm, 20 *10 cm, and 20 *20 cm.
• Only 5 ml of solvent is required per trough for HPTLC plate in a
10 10 cm chamber.
2. Automatic Developing Chamber (ADC)
Here there is isocratic development of HPTLC plates.
Steps like preconditioning of layer, chamber saturation,
development distance and final drying can be preset and
automatically monitored independent of environmental effects .

32. Reference

33. Reference
• P.Bedson and M.Sargent, The development and application of guidance on
equipment qualification of analytical instruments, Accreditation and
Quality Assurance, 1 (6), 265/274 (1996).
• L. Huber, Validation and qualification in analytical laboratories, Text
book, 300 pages, 1998, Interpharm, Bufallo Grove, IL, USA, Nov. 1998
(book).
• L. Huber, Equipment Qualification in Practice LC/GC Magazine, Vol
16,February1998,148-156
• book).
• Ph.Eur.2.2.35 chromatography , High performance liquid chromatography
• Guidance on equipment qualification of analytical instruments
• Journal of Perkin Elmer life & analytical science