HPLC is characterized by using high pressure to push a mobile phase through a stationary phase column, allowing for the separation of complex mixtures. Validation of analytical methods demonstrates that the methods are accurate, precise, specific, sensitive, linear, and robust for their intended purpose. Key aspects of validation include accuracy, precision, specificity, detection limit, quantitation limit, linearity, range, and robustness. Validation ensures methods provide reliable results.

1. HPLC
Ahsan Saqi
The Islamia University of Bahawalpur

2. High Performance Liquid Chromatography
HPLC is characterized by the use of high pressure to
push a mobile phase solution through a column of
stationary phase allowing separation of complex
mixtures with high resolution

3. Chromato-graphy / -graph / -gram / -grapher
• Chromatography: Analytical technique
• Chromatograph: Instrument
• Chromatogram: Obtained “picture”
• Chromatographer: Person

4. Absorption:
In chromatography, absorption signifies the process by which a solute
partitions into a liquid-like stationary phase.
Adsorption:
The process by which a chemical entity is accumulated on a surface.
Mobile Phase:
The eluate moving through the column. In gas chromatography (GC)
this will be a gas, and in liquid chromatography (LC) a liquid.
Stationary Phase:
The substance that remains in one place in the column. In GC this will
be a liquid of high-viscosity, which clings to the inner walls of the
column; in LC it will be some sort of packing, either solid or gel-based.

5. Capillary Column:
A column whose inner diameter is under 0.5 mm.
Eluate:
The mobile phase exiting a column.
Eluent:
The mobile phase entering a column.
Elution:
The passage of the mobile phase through the
column to transport solutes

6. Partition Chromatography:
A type of chromatography based on a thin film formed on the
surface of a solid support by a liquid stationary phase. Solute
equilibrates between the mobile phase and the stationary liquid.
Flow Rate:
The amount of mobile phase that has passed through the column
per unit time. The units are milliliters per second (mL/sec) or,
more commonly, milliliters per minute (mL/min).
Solute:
The term for the sample components being analyzed.

8. HPLC System
Solvent
Delivery System
Variable
UV/Vis Detector
HPLC Solvent
Reservoirs
HPLC
Column
Injector
Detector Computer
Workstation
Keep an eye on
these 4 screens!

9. Solvent Delivery System

10. Solvent Delivery System
Injector
%A %B %C Flow Rate Pressure
{H2O} {MeOH} (mL/min) (atmos.)
Ready
Ternary Pump
A
C
B
from solvent
reservoir
Column
to
detector
to column
through
pulse
dampener
to injector
through pump
load
inject

11. UV/Visble Detector
ABS Flow rate l RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min
Ready

12. HPLC Working

13. Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic column
chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Degasser
Flow Channel Diagram for High Performance
Liquid Chromatograph

14. Picture of an HPLC column

15. HPLC Column
• Material
– Stainless steel (SUS)
– PEEK (polyether ether
ketone)
– Fluororesin
• O.D. (outer diameter)
– 1.6 mm
• I.D. (inner diameter)
– 0.1 mm
– 0.3 mm
– 0.5 mm
– 0.8 mm etc.

16. Column Storage
• Storage Solution
– It is generally safe to use
the same storage solution
as used at shipment.
– In order to prevent
putrefaction, alcohol or
some other preservative
substance may be added.
• Storage Conditions
– Insert an airtight stopper in the
column end.
– Never allow the packing
material to dry.
– Make a record of the storage
solution and final usage
conditions and store it together
with the column.
– Store the column in a location
not subject to shocks or sudden
temperature changes.

17. WHAT will AFFECTS the SYSTEM?
Column
Parameters
• Column Material
• Deactivation
• Stationary Phase
• Coating Material
Instrument
Parameters
• Temperature
• Flow
• Signal
• Sample Sensitivity
• Detector
Sample
Parameters
• Concentration
• Matrix
• Solvent Effect
• Sample Effect

19. HPLC Chromatogram
tR
t0
Intensityofdetectorsignal
Time
Peak
tR : Retention time
h
A
t0 : Non-retention time
A : Peak area
h : Peak height

20. HPLC Chromatograms
Rt = 3.0 min.
faster moving
less retained
Rt = 5.2 min.
slower moving
more retained
0 1 2 3 4 5 6 7
Time (minutes)
Absorbance
Approximation
of peak area by
triangulation
Area =
base x height
2
base
height
Peak A Peak B

21. Normal phase
• In this column type, the retention is governed
by the interaction of the polar parts of the
stationary phase and solute. For retention to
occur in normal phase, the packing must be
more polar than the mobile phase with respect
to the sample

22. Reverse phase
• In this column the packing material is relatively
nonpolar and the solvent is polar with respect to the
sample. Retention is the result of the interaction of
the nonpolar components of the solutes and the
nonpolar stationary phase. Typical stationary phases
are nonpolar hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the solvents
are polar aqueous-organic mixtures such as methanol-
water or acetonitrile-water.

23. Normal vs. Reversed Phase Chromatography
Normal Phase Reversed Phase
Stationary phase Polar (silica gel) Non-polar (C18)
Mobile phase
Non-polar
(organic solvents)
Polar
(aqueous/organic)
Sample movement Non-polar fastest Polar fastest
Separation based on
Different polarities
(functionality)
Different
hydrocarbon content

24. Chromatography Stationary Phases
relatively polar surface
O O O
| | |
OSiOSiOSiOH
| | |
O O O
| | |
OSiOSiOSiOH
| | |
O O O
bulk (SiO2)x surface
relatively nonpolar surface
Silica Gel
O O O
| | |
OSiOSiOSiOR
| | |
O O O
| | |
OSiOSiOSiOR
| | |
O O O
bulk (SiO2)x surface
Derivatized Silica Gel
Where R = C18H37
hydrocarbon chain
(octadecylsilyl deriv.
silica or “C18”)
“normal phase” “reversed phase”

26. Validation:
A programe which guarantees the accuracy, specificity, precision
and robustness of a method or process.

27. Validation of method
• Scientifically demonstrating
that the analytical methods
concur with the intended
purpose (i.e., that errors are
within a permissible range)
• Evaluating required items
from the validation
characteristics
• Validation characteristics
– Accuracy / trueness
– Precision
– Specificity
– Detection limit
– Quantitation limit
– Linearity
– Range
– (Robustness)

28. Accuracy / Trueness
• Definition
– Degree of bias in
measurements obtained with
analytical procedures
– Difference between true
value and grand mean of
measurements
• Evaluation Method
– Comparison with theoretical
values (or authenticated
values)
– Comparison with results
obtained using other
analytical procedures for
which the accuracy
(trueness) is known
– Recovery test

29. Precision
• Definition
– Degree of coincidence of
series of measurements
obtained by repeatedly
analyzing multiple samples
taken from a homogenous
test substance
– Variance, standard deviation,
or relative standard deviation
of measurements
• Repeatability / Intra-Assay
Precision
– Precision of measurements
taken over a short time
period under the same
conditions
• Intermediate Precision
• Reproducibility

30. Specificity
• Definition
– The ability to accurately
analyze the target substance in
the presence of other expected
substances
– The discrimination capability
of the analytical methods
– Multiple analytical procedures
may be combined in order to
attain the required level of
discrimination
• Evaluation Method
– Confirmation that the target
substance can be
discriminated (separated)
from co-existing components,
related substances,
decomposition products, etc.
– If reference standards for
impurities cannot be obtained,
the measurement results for
samples thought to contain
the impurities are compared.

31. Detection Limit
• Definition
– The minimum quantity of a
target substance that can be
detected.
– Quantitation is not
absolutely necessary.
• Evaluation Method
– Calculated from the standard
deviation of measurements
and the slope of the
calibration curve.
• DL = 3.3 /slope
(: Standard deviation of
measurements)
(Slope: Slope of calibration
curve)
– Calculated from the signal-
to-noise ratio.
• Concentration for which S/N =
3 or 2

32. Quantitation Limit
• Definition
– The minimum quantity of a
target substance that can be
quantified
– Quantitation with an
appropriate level of
accuracy and precision
must be possible. (In
general, the relative
standard deviation must not
exceed 10%.)
• Evaluation Method
– Calculated from the standard
deviation of measurements and
the slope of the calibration curve.
• QL = 10 /slope
(: Standard deviation of
measurements)
(Slope: Slope of calibration curve)
– Calculated from the signal-to-
noise ratio.
• Concentration for which S/N = 10

33. Linearity
• Definition
– The ability of the analytical
method to produce
measurements for the
quantity of a target
substance that satisfy a
linear relationship.
– Values produced by
converting quantities or
measurements of the target
substance using a precisely
defined formula may be
used.
• Evaluation Method
– Samples containing different
quantities of the target
substance (usually 5
concentrations) are analyzed
repeatedly, and regression
equations and correlation
coefficients are obtained.
– Residuals obtained from the
regression equations of the
measurements are plotted,
and it is confirmed that there
is no specific slope.

34. Range
• Definition
– The region between the
lower and upper limits of the
quantity of a target
substance that gives
appropriate levels of
accuracy and precision
• Evaluation Method
– The accuracy, precision, and
linearity are investigated for
samples containing
quantities of a target
substance that correspond to
the lower limit, upper limit,
and approximate center of
the range.

35. Robustness
• Definition
– The ability of an analytical
procedure to remain
unaffected by small
changes in analytical
conditions.
• Evaluation Method
– Some or all of the variable
factors (i.e., the analytical
conditions) are changed
and the effects are
evaluated.

36. Advantages of methods validation:
There are a number of very good reasons why
analytical methods are validated. First, the laboratory
conducting the tests will have an increased output with
fewer reductions in rejections of the results. The
method is therefore cost effective and avoids additional
capital expenditure on other equipment and reduced
testing of the finished goods e.g. a batch of drugs.