QUALIFICATION OF HPLC , HPLC

QUALIFICATION OF HPLC
PRESENTED BY ,
SURIYAPRIYA .K
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
KMCH COLLEGE OF PHARMACY
COIMBATORE
kmch college of pharmacy 1

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)

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.
• Sample interacted little with column material elute quickly and are characterized by short
retention time.
• Retention time of analyte depends on sample, solvent, nature of stationery phase .

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.

INSTALLATION QUALIFICATION :
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.

BEFORE
INSTALLATION
Obtain manufactures
recommendations for
installation
Check the site for fulfilment for
utilities such as electricity
,temperature etc
Allow sufficient space for equipment,SOPs,
operating manual and software.

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

OPERATIONAL 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.

VERIFICATION REPORT

BASELINE NOISE AND DRIFT:
• Drift and baseline noise are important factors .
• Increased baseline noise considerably reduces the sensitivity, as it is not possible to
distinguish between low-level signals and noise.
• With increased drift, it is more difficult to integrate the signals correctly because the
less stable the baseline is, the more inaccurate is integration.

CONTINUED
• The baseline noise of the detector mainly depends on the lamp.
• There is a considerable increase in noise,
If an old lamp with poor light intensity is used.
When the flow cells is dirty.
Flow cells should free from gas bubbles.
• To measure the drift of a UV detector, also make sure that all measuring conditions are
constant
• The lamp intensity decreases while the lamp is burning.
• when use turned on and off often.

EVALUATING BASELINE NOISE AND DRIFT :
• To check noise, drift water is pumped through the cell at a flow rate of 1ml/min. The
UV signal is recorded at 254nm.
• To calculate noise the measuring signal is split into 20 intervals for 1min each.
• For each interval chromeleon calculates a regression based on measured values,
using the method of least square.
• The limit should be between <2 x 10- 3 AU. To calculate the drift, chromeleon
calculates a regression line from all data points with in a range of 1-21mins based
on the method of least square.
• The slope of the regression line is the calculated drift.
• The limit should be between <5 x 10—5 AU.

PRECISION OF INJECTION VOLUME :
Precision of injection volume is an important parameter for accuracy of quantitation.
EVALUATING PRECISION OF INJECTION VOLUME
Inject 6 standard caffeine solution and calculate height, area, average height, average area,
%RSD of height and %RSD of area which gives precision of volume and the limit should be in
between 0.3% RSD.
DETECTOR LINEARITY :
It is a critical parameter to establish for reliable and accurate quantitative results.
EVALUATING DETECTOR LINEARITY: A series of 5 traceable standards (caffeine soln of
con. about 0.00035 to 0.35mg/ml) are injected and evaluated. The detector linearity is
calculated by determining the peak area vs concentration. The limit should be in between >1.5
AU, 5% RSD.

WAVELENGTH ACCURACY :
It is an important parameter for accuracy of quantitative
and qualitative analysis.
EVALUATING WAVELENGTH ACCURACY :
• Traceable caffeine standard is used to determine the
wavelength accuracy.
• Caffeine is trapped in the flow cell and a programmable
timetable is used to determine the wavelength maxima (205nm) and
minima (273nm).
• The wavelength accuracy is determined as the absolute
difference between the measured and certified wavelength values.

TEMPERATURE ACCURACY :
Temperature fluctuations of the solvent and column can result in retention time
fluctuations.
Therefore, accuracy of the temperature is important.
EVALUATING TEMPERATURE ACCURACY:
• Four measuring points are used to check temperature accuracy of column.
• The temperature achieved is measured with external calibrated thermometer.
• The achieved temperatures are compared to the set values.
• The difference indicates the temperature accuracy and the limit should be in between ± 1º c

TEMPERATURE PRECISION :
Monitor temperature for 20 min ,and limit should be in between ±0.25c .
AUTO SAMPLER CARRY OVER :
• Once the sample is highly concentrated ,a sample containing only solvent is injected.
• Ideally only the signal for the solvent is displayed in the chromatogram.
• If a signal for the sample is displayed, this indicates the carry over by the
autosampler.
EVALUATING AUTOSAMPLER CARRY OVER:
Run the sample containing only solvent. The signal for solvent will be displayed . If other
signals are displayed it is due to auto sample carry over. Should be less than 0.5%

DOCUMENTATION:
On completion of equipment qualification, documentation should be available that
consists of :
 Design qualification document .
 IQ document (includes description of hardware and software)
 Procedures for OQ testing.
 OQ test reports (includes test parameters, acceptance criteria & actual results ).
 PQ test procedures and representative results.

REFERENCE
 Ph.Eur.2.2.35 chromatography; High performance liquid
chromatography
 Guidance on equipment qualification of analytical instruments
 Journal of Perkin Elmer life & analytical science
 https://www.slideshare.net/huzaifanaaz/qualification-of-high-
performance-liquid-chromatography

QUALIFICATION OF HPLC , HPLC

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QUALIFICATION OF HPLC , HPLC