PERFORMANCE VERIFICATION PRACTICES

Pump Module
Flow Rate Accuracy:
 The flow-rate accuracy of the pump can be evaluated simply by calculating the
time required to collect a predetermined volume of mobile phase at different
flow rate settings.
 For example, the flow-rate accuracy at 2 mL/min can be verified by using a calibrated
stopwatch to measure the time it takes to collect 25 mL of effluent from the pump into a
25-mL volumetric flask. A calibrated flow meter can be used to determine the flow rate
as well.
 A calibrated flow meter can be used to determine the flow rate as well.. A steady
backpressure may be required, depending on the requirement of the system.

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Gradient Accuracy and Precision:
• The gradient operation precision can be assessed indirectly by monitoring the
relative standard deviation in retention time of peaks in the chromatographs from
repeated injections.
• The accuracy and linearity of the gradient solvent delivery can be verified
indirectly by monitoring the absorbance change when the composition of the two
solvents from two different channels changes.
• High-pressure gradient runs usually involve two solvent systems. Lower-
pressure gradient LC pumps are usually equipped with quaternary proportioning
valves, which can handle up to four solvents. The test will be performed for two
channels at a time.

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 Pump Module
Pressure Test:
The performance of the LC pump depends on the proper
functioning of the pump seal, check valves, and proper
connection of the tubing. Properly functioning pump seal check
valves and tubing connections are important in maintaining stable
mobile-phase flow and system pressure.

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 Pump Module
Pressure Test:
When performing the pump pressure test, make sure that the
system is well primed so that no bubbles are present in the pump
system. The pressure fluctuation caused by the presence of air
bubbles in the pump will lead to misinterpretation of the test
results.

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 Pump Module
Pressure Test:
The first step in the pressure test is to plug the outlet of the pump
using a dead-nut. The automatic pump shutdown pressure is set
to 4000 psi. The pump head pressure signal output is connected
to a recorder (this test can be performed without using a
recording device by visually monitoring the pressure reading as
the system is being pressurized).

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 Pump Module
Pressure Test:
Pressurize the pump by pumping methanol at 1 mL/min.
Methanol is less viscous than water and is more sensitive to leaks
in the system. The pressure inside the pump head increases
quickly as the outlet of the pump is blocked. As the pressure
increases to about 3000 psi, the flow rate is reduced to 0.1
mL/min.

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Pump Module

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 Pump Module
Pressure Test:
The pressure in the pump head decreases slowly over time after
the automatic shutdown. Typically, the pressure drop is less than
10% over 5 min. A steep decrease in pressure over time implies
poor check-valve performance or leaks within the pumping
system.

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 Injector Module
Precision:
The ability of the injector to draw the same amount of sample in
replicate injections is crucial to the precision and accuracy for
peak-area or peak height comparison for external standard
quantitation. If the variability of the sample and standard being
injected into the column is not controlled tightly, the basic
principle of external standard quantitation is seriously
compromised.

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 Injector Module
Precision:
No meaningful comparison between the responses of the sample
and the standard can be made. The absolute accuracy of the
injection volume is not critical as long as the same amount of
standard and sample is injected.

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 Injector Module
Precision:
The volume precision of the injector can be demonstrated by
making at least six replicate injections from a sample. The relative
standard deviation (% RSD) of the response of the injections is
then calculated to evaluate the precision. A fast and sample
analysis should be considered for the test. The run condition for a
very simple LC analysis of caffeine is given below as an example.

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 Injector Module
Precision:

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 Injector Module

Linearity:
Most automated LC injectors are capable of varying the injection
volume. A variable volume of sample will be drawn into a sample
injection loop by a syringe or other metering device. The
uniformity of the sample loop and the ability of the metering
device to draw different amounts of sample in proper proportion
will affect the linearity of the injection volume.

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 Injector Module
Linearity:
Linearity is important for methods that require the use of variable
injection volumes, such as the high–low method in the
quantitation of impurities (see Chapter 3 for the validation of
related substances method). The linearity of the injector can be
demonstrated by making injections, typically 5,10, 20, 50, and
100 μL, to cover the range 0 to 100 μL.

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 Injector Module
Linearity:
The response of the injection is plotted against the injection
volume. The correlation coefficient of the plot is used in
evaluation of the injection linearity. Carryover:

Small amounts of analyte may get carried over from the

previous injection and contaminate the next sample to be
injected [10]. The carryover will affect the accurate quantitation
of the subsequent sample. The problem is more serious when a
dilute sample is injected after a concentrated sample

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 Injector Module
Carryover:
To avoid cross-contamination from the preceding sample
injection, all the parts in the injector that come into contact with
the sample (the injection loop, the injection needle, and the
needle seat) have to be cleaned effectively after the injection. The
carryover can be evaluated by injecting a blank after a sample
that contains a high concentration of analyte.

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 Injector Module

Carryover:
The response of the analyte found in the blank sample expressed
as a percentage of the response of the concentrated sample can be
used to determine the level of carryover. Caffeine can be used for
the system carryover test for assessing the performance of an
injector and serves as a common standard for comparing the
performance of different injectors.

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 Resolution

• Insufficient resolution leads to a decrease in the extinction

coefficient across the wavelength axis, and therefore inaccurate
quantitation results.
• The resolution of a UV–Vis spectrophotometer is related to its
spectral bandwidth (SBW).
• The smaller the spectral bandwidth, the finer the resolution.
• The SBW depends on the slit width and the dispersive power of
the monochrometer.
 Resolution

• The resolution of the absorbance measurement depends on the

ratio of the spectral bandwidth (SBW) of the spectrophotometer to
the natural bandwidth (NBW) of the spectral band to be measured.
The NBW is a physical characteristic of the analyte of interest.
• The accuracy is not likely to be affected if the ratio of NBW/SBW
is greater that 20. If the ratio is less than 10 (SBW increasing), the
measured spectrum becomes distorted.
 Resolution

• Tests. A solution mixture of 0.02% v/v toluene in hexane (UV

grade) is used to test the resolution power of the
spectrophotometer. Hexane as the reference is scanned and
then the spectrum of the resolution solution from 250 to 300
nm is obtained. The absorbance values of the λmax at 269 nm
and the λmin at 266 nm are recorded (Figure 10.7).
• Acceptance: The ratio of the absorbance at λmax (269 nm) and
absorbance at λmin (266 nm) should be greater than 1.5.
 Noise

• Noise in the UV–Vis measurement originates primarily from the light

source and electronic components. Noise in the measurement affects the
accuracy at both ends of the absorbance scale.
• Photon noise from the light source affects the accuracy of the
measurements at low absorbance.
• Electronic noise from the electronic components affects the accuracy of
the measurements at high absorbance.
• A high noise level affects the precision of the measurements and reduces
the limit of detection, thereby rendering the instrument less sensitive.
Noise
 Noise

• Tests. Air is scanned in the absorbance mode for 10 min. peak-to-peak

noise is recorded at 500 nm. The root mean square (RMS) noise is then
calculated. The RMS noise measurement is a measure of the standard
deviation of the background signals. Modern spectrophotometers are
usually equipped with the noise estimation function. For older
spectrophotometers, the RMS noise can be estimated by multiplying
the highest peak-to-peak noise level by a factor of 0.7 (Figure 10.8).
• Acceptance: The RMS noise should typically be less than 0.001 AU.
 Wavelength Accuracy

• Wavelength accuracy is defined as the deviation of the wavelength reading at

an absorption band or emission band from the known wavelength of the band.
• The true λmax and λmin of the analyte cannot be characterized accurately.
• Wavelength accuracy verification is checked by measuring a known wavelength
reference standard with well-characterized absorption or emission peaks and
comparing the recorded wavelength of the peak(s) against the value(s) listed in
the certificate of that reference standard.
• Spectra of some commonly used wavelength standards such as a deuterium
lamp, mercury vapor lamp, holmium oxide filter, and holmium oxide solution
(4% holmium oxide in 10% perchloric acid in a 1-cm cell) are illustrated in
Figure 10.3a–d, respectively.
 Wavelength Accuracy

• Holmium oxide solution is commercially available in a sealed 1-cm cuvette. It

is a very convenient and versatile wavelength standard.
• The standard is suitable for UV–Vis spectrophotometers typically used in
pharmaceutical laboratories, with spectral bandwidth ranging from 2 to 0.5 nm.
• The certified wavelength values of the peaks are listed in Table 10.3. When a
spectral bandwidth of 0.5 nm or narrower is used in a measurement, the
position of peak number 10 at about 451 nm becomes undefined, due to peak
splitting as a result of enhanced resolution.

Acceptance:
• ±1 nm in the UV range (200 to 380 nm) and ±3 nm in the visible range (380 to
800 nm). Three repeated scans of the same peak should be within ±0.5 nm.
Wavelength Accuracy
 Stray Light

• Stray light is defined as the detected light of any wavelength that is

outside the bandwidth of the wavelength selected.
• The causes for stray light are scattering, higher-order diffraction of the
monochromator, or poor instrument design.
• Stray light causes a decrease in absorbance and reduces the linear range
of the instrument.
• High-absorbance measurements are affected more severely by stray light.
For example, consider the following measurement scenarios at 1%
transmittance level:
Stray Light
 Stray Light

• The stray light problem causes a deviation from linearity at

high absorbance. In general, the linearity of the absorbance
response is limited by stray light at high absorbance and by
noise at the low absorbance range. Absorbance values ranging
from 0.3 to 1 AU are less susceptible to stray light and noise
problems and therefore become the preferred absorbance
range for UV–Vis analyses.
Stray Light