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International Journal of Chromatographic Science

Universal Research Publications. All rights reserved

Original Article
Performance Verification Test of High Performance Liquid Chromatography:
Practical Example
Lantider Kassaye, Getachew Genete
Food, Medicine and Health Care Administration and Control Authority, Food and Drug quality Control Laboratory, Addis
Ababa, Ethiopia
Received 18 January 2013; accepted 11 March 2013
Abstract
The performance of an HPLC system can be evaluated by examining the key functions of the various modules that
comprise the system. The pump, auto injector, column oven and the detector are the most important parts of an HPLC
system which needs to be verified for the proper functioning. Flow rate accuracy and gradient accuracy for the pump;
Precision, Linearity and carry over for the auto sampler; wavelength accuracy and response linearity for the detector and
temperature accuracy for the column oven are the most important parameters which need to be considered during HPLC
performance verification. In this practical example, all of the modules except column oven (for more than 50 oC) meet the
acceptance/refusal criteria suggested by Herman Lam.
© 2013 Universal Research Publications. All rights reserved
Key words: Verification of HPLC; HPLC; Performance qualification of HPLC
Introduction known values of measured quantity are under control.
Good analytical results are essential in order to take reliable High-Performance Liquid Chromatography (HPLC) is one
decisions. Analytical measurements affect the daily lives of of the foremost analytical techniques widely used in
every citizen. Sound, accurate and reliable analytical analytical laboratories for the analysis of pharmaceuticals
measurements are fundamental to the functioning of and chemicals [6-8], foods [9 -11], cosmetics samples and
modern society. A wrong result can have an enormous so on [12, 13]. In order to provide a high level of assurance
social and economic impact [1]. The correctness of that the data generated from the HPLC analysis are reliable,
measurements and measuring instruments is one of the key the performance of the HPLC system should be monitored
prerequisites to ensure the quality of products and services, at regular intervals.
and the accuracy of the instruments must be consistent with The performance of an HPLC system can be evaluated by
their intended use [2]. examining the key functions of the various modules that
Calibration and verification are the most important actions comprise the system. The common HPLC performance
to ensure the correct indication of measuring instruments attributes and the acceptance/refusal criteria are presented
[2]. Regular calibration of measuring instruments should be in Table 1. The acceptance/refusal criteria values for these
carried out in agreement with the implemented quality attributes are based on the values suggested by Herman
systems. The industrial metrology ensures the appropriate Lam in the chapter “Performance Verification of the
functioning of measurement instruments used in industry as HPLC” of his book [14].
well in production and testing processes, in order to 2. Experimental
guarantee the quality of life for citizens and for academic 2.1 Instruments and apparatuses
research [3]. A low pressure quaternary HPLC instrument comprises
Verification is the confirmation, based on evidences (facts, LC-10ATvp pump, SIL-10ADvp auto sampler, SPD-
test results) that some specified requirement has been 10AVvp UV-Vis detector and CTO-10AVP column oven
fulfilled. The result from a verification assay will show if (Shimadzu, Japan) has been verified for its proper
the measuring equipment is in agreement with its required functioning. Analytical balance (METTLER Toledo,
specifications, which are generally expressed as tolerances Switzerland), Calibrated stop watch (Treaceble®
[4]. The verification of measuring instruments includes stopwatch, JUMBO DIGIT, VWR, China) and
testing and requires the availability of clear specifications thermometer (Service Testo, Germany) have been used.
and acceptance/refusal criteria [5]. Verification provides 2.2 Materials and chemicals
means of checking that the deviations between the values ODS analytical column (150 X 4.6 mm, 5μm, Waters,
displayed by a measuring instrument and the corresponding USA), Class A 25mL volumetric flasks (Pyrex, Germany),

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18
methanol HPLC grade (BDH, USA), Acetonitrile HPLC in methanol). The relative standard deviation (RSD) of the
grade (J.T. Baker, USA) and caffeine (Fluka, Sigma- response of the injections was calculated to evaluate the
Aldrich, China, purity ≥ 0.99%) were employed for this precision.
work. Linearity
Table 1: Performance attributes and their acceptance/ The linearity of the injector was demonstrated by making
refusal criteria for HPLC verification injections of 5, 10, 20, 30 and 50 μL of the caffeine
Performance Acceptance/refusal solution (0.05 mg/mL in methanol). The response of the
Module injections at each injection volume was plotted against the
Attributes criteria
± 1% of the set injection volume. The correlation coefficient of the plot
Pump Flow rate accuracy was used in the evaluation of the injection linearity.
flow rate
± 1% of the step Carryover
Carryover was evaluated by injecting a blank (methanol)
Gradient accuracy gradient
after a sample that contains a high concentration of analyte
composition
Auto injector Precision RSD ≤ 1% ((0.2 mg/mL of caffeine in methanol). The response of the
Linearity r ≥ 0.999 analyte found in the blank sample expressed as a
percentage of the response of the concentrated sample was
Carryover < 1%
used to determine the level of carryover.
Wave length
Detector ± 2nm 2.3.2.3. UV-Visible Detector Module:
accuracy
Linearity of Wavelength Accuracy
r ≥ 0.999 The wavelength verification of the UV range was
response
Column Temperature ± 2 oC of the set performed by filling a flow cell with a
Caffeine solution (0.015mg/mL in methanol) and the UV
oven accuracy temperature
spectrum was collected in the range of 200 to 400nm. The
2.3 Methods obtained scan should have λmax at about 272nm and λmin at
2.3.1 Chromatographic condition (For Auto sampler about 244nm.
and Detector verification) Linearity of Response
A mobile phase containing 85% water and 15% acetonitrile The linearity of the detector response was checked by
was pumped through the analytical column (ODS, 150 X injecting a series of standard solutions of caffeine (0.05,
4.6mm, 5μm) at a flow rate of 2 mL/minute. The test 0.1, 0.2, 0.4 and 1 mg/mL) to the chromatographic system.
solutions, caffeine in methanol, were injected and detected From the plot of response versus the concentration of the
at 272nm. The column temperature was ambient. solutions, the correlation coefficient between sample
2.3.2 Verification Test for different modules of HPLC concentration and response was calculated to determine the
2.3.2.1 Pump Module: linearity.
Flow Rate Accuracy 2.3.2.4. Column Oven module:
The flow-rate of the pump was set at 2 mL/min. and the The accuracy of the oven was checked at 30, 40, 50, 60 and
time required to fill a 25 mL volumetric flask was 70 oC using the calibrated thermometer. The correction
measured using the calibrated stopwatch. factors obtained from the calibration certificate of the
Gradient Accuracy and Linearity thermometer were considered to get the experimental
The accuracy and linearity of the gradient solvent delivery temperature.
has been verified indirectly by monitoring the absorbance Results and Discussion
change as the binary composition of the two solvents 1. Pump module
changes from two different channels. The LC gradient has 1.1. Flow rate Accuracy
four channels: A, B, C and D. The test was performed for One of the key performance requirements for the pump
two channels at a time. Channel A is filled with a pure module is the ability to maintain accurate and consistent
solvent, methanol, while channel B is filled with a solvent flow of the mobile phase, which will be necessary to
containing a UV-active tracer, caffeine. The gradient provide stable and repeatable interactions between the
profile is programmed to pump from solvent A only for two analytes and the stationary phase [15]. Poor flow-rate
minutes and then to decrease the composition of Solvent A accuracy will affect the retention time of the separation. As
by 10% and to increase solvent B by the same percent and shown in table 2, the results for flow rate accuracy for all of
allowing to pump for two minutes at each change. The the four suction channels is in the range of 99.04 to 100.4
absorbance change at each composition change is measured % and this good result indicates that the pump performance
and expressed as height H in the plot of absorbance versus is superior.
solvent composition. The linearity of the gradient delivery 1.2. Gradient Accuracy and Linearity
was verified by plotting the absorbance at various mobile For gradient accuracy, the ability of the pump to deliver the
phase compositions versus the theoretical composition. The mobile phase at different solvent strengths over the time by
entire process was repeated for channels C and D. varying the composition of the mobile phase accurately is
2.3.2.2 Injector Module: fundamental to get the adequate chromatographic
Precision separation and reproducibility.
The precision of the injector was demonstrated by making As shown in fig 1, the absorbance increased step by step as
six replicate injections from a sample (caffeine 0.05 mg/mL the composition of the caffeine increased. Similarly, the

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19
absorbance decreased to zero as the composition of the caffeine and the obtained height is linear with a correlation
caffeine decreased to zero. The height increased due to the coefficient of 0.9998. Table 3 and Table 4, shows the
change in caffeine composition was measured by the LC result for gradient accuracy for channel A and B and
solution software from the baseline and these heights have channel C and D respectively. The accuracy is calculated
been used for the determination of gradient accuracy and by determining the response factor for each percentage
gradient linearity. The gradient linearity is determined by change and comparing it with the average response factor.
graphing the percentage composition of caffeine versus the The obtained accuracies at each level are in agreement with
respective obtained height. As shown in figure 2, the the acceptance/refusal criteria for all channels.
relationship between percentage of the composition of
Table 2: Flow rate accuracy test result the four pump channels separately.
a
Time directly measured by the stopwatch
b
Time converted to minutes
Line A Line B Line C Line D
a
Vol. Theoretical Time b Accu a b Accur a b Accur a b Accur
Rep Time Time Time Time Time Time Time
(mL) time (min) (min:Sec racy acy acy acy
(min) (min:Sec) (min) (min:Sec) (min) (min:Sec) (min)
) (%) (%) (%) (%)
1 25 12.5 12:26 12.43 99.44 12:30 12.5 100.00 12:33 12.55 100.40 12:28 12.47 99.76
2 25 12.5 12:25 12.42 99.36 12;29 12.48 99.84 12:30 12.5 100.00 12:26 12.43 99.44
3 25 12.5 12:26 12.43 99.44 12:28 12.47 99.76 12:29 12.48 99.84 12:27 12.45 99.60
4 25 12.5 12:27 12.45 99.60 12:27 12.45 99.60 12:27 12.45 99.60 12:27 12.45 99.60
5 25 12.5 12:26 12.43 99.44 12:23 12.38 99.04 12:28 12.47 99.76 12:27 12.45 99.60
Table 3: Gradient accuracy results for channel A and B.
a b
Channel Channel Time Height 1 Height 2 Height 3 Average Response Acc.
A (%) B (%) (min) (Abs Units) (Abs Units) (Abs Units) (Abs Units) Factor (%)
100 0 2 0 0 0 0 NA
90 10 4 69506 70058 70356 69973 6997.3 98.6
80 20 6 145847 144592 149227 146555 7327.8 100.4
70 30 8 221595 218145 220563 220101 7336.7 100.5
60 40 10 292574 292520 293820 292971 7324.3 100.3
50 50 12 368022 370379 368457 368953 7379.1 101.1
40 60 14 438919 438899 438377 438732 7312.2 100.2
30 70 16 512502 514026 513741 513423 7334.6 100.5
20 80 18 583598 584747 591672 586672 7333.4 100.5
10 90 20 661655 660386 659681 660574 7339.7 100.5
0 100 22 733540 730582 731038 731720 7317.2 100.2
Mean 7300.2 100.3
a
Response factor = Average height/percentage composition of caffeine
b
Accuracy (%) = Response factor* 100/Average response factor
Table 4: Gradient accuracy results for Channel C and D.
a
Response factor = Average height/percentage composition of caffeine
b
Accuracy (%) = Response factor* 100/Average response factor
a b
Channel C Channel Time Height 1 Height 2 Height 2 Ave. Height Response Acc.
(%) B (%) (min) (Abs Units) (Abs Units) (Abs Units) (Abs Units) Factor (%)
100 0 2 0 0 0 0 NA
90 10 4 70567 72446 72635 71883 7188.3 98.7
80 20 6 143868 148397 150494 147586 7379.3 100.6
70 30 8 216505 219043 221376 218975 7299.2 99.5
60 40 10 292260 295224 303983 297156 7428.9 101.3
50 50 12 363730 368817 369963 367503 7350.1 100.2
40 60 14 437677 441136 443041 440618 7343.6 100.2
30 70 16 511447 514552 520466 515488 7364.1 100.4
20 80 18 581062 586289 587411 584921 7311.5 99.7
10 90 20 659430 661308 664928 661889 7354.3 100.3
0 100 22 729972 730282 731290 730515 7305.1 99.6
Mean 7332.4 100.1
2. Auto injector module external standard quantitation. If the variability of the
2.1. Precision sample and standard being injected into the column is not
The ability of the injector to draw the same amount of controlled tightly, the basic principle of external standard
sample in replicate injections is crucial to the precision and quantitation is seriously compromised. No meaningful
accuracy for peak-area or peak-height comparison for comparison between the responses of the sample and the

International Journal of Chromatographic Science 2013, 3(1): 18-23

20
standard can be made. The absolute accuracy of the Table 5: Result for the auto sampler precision
injection volume is not critical as long as the same amount Replicate Peak area
of standard and sample is injected. Table 5 shows the result
1 1098235
for the auto sampler precision test and it proves that the
auto sampler is precise as the relative standard deviation for 2 1101507
the six injections is less than 1%. 3 1097638
4 1098576
5 1103366
6 1061430
Average 1093459
STDVE 15844
RSD 0.23
2.2 Linearity
Most of the automated LC injectors are capable of varying
the injection volume without changing the injection loop.
Variable volume of sample will be drawn into a sample
Fig 1: Chromatogram for gradient accuracy test of channel
injection loop by a syringe or other metering device. The
A and B
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.
Most of the employed volumes of injection in HPLC are in
the range of 5 to 100 µL. However, in this verification test,
the auto sampler linearity has been checked at maximum of
50µL which is the installed loop size for the instrument. As
table 6 and fig 3 shows, there is good relationship between
volume of injection and response (peak area). The
Fig 2: Graph which shows the relationship between correlation coefficient is 0.999998 which is more than the
percentage composition of caffeine and height for channel acceptance/refusal criteria, 0.999.
A and B
Table 6: Results for Auto injector linearity with its acceptance/refusal criteria
Inj. vol (µL) Peak Area 1 Peak area 2 Peak area 3 Average RSD
5 533967 531499 533654 533040 0.252081
10 1063166 1064724 1067951 1065280 0.229097
20 2105973 2113965 2114930 2111623 0.232830
50 5269317 5282999 5306851 5286389 0.359324
Correlation coefficient, r 0.999998
Accepted/Not Accepted Accepted
calculating the ratio between the responses (peaks-area) of
the caffeine found in the methanol sample and the standard
solution.
As shown in Table 7, the average percent carry over (n=3)
for the auto sampler is 0.26% which is much lower than the
acceptance/refusal criteria, ≤ 1%. The chromatogram for
the sample and blank are shown in fig. 4 and 5.
And hence we can say that the cleansing procedure for the
auto sampler is good. Moreover, results for precision and
linearity prove that the auto sampler is in good condition to
Fig 3: Graph for the relationship between volume of use it for quantitative analysis.
injection and peak area Table 7: Carry over test results for the auto injector
2.3. Carry over Carry Accepted/Not
Small amounts of analyte may get carried over from the Replicate Peak Area
over (%) Accepted
sample injected before and lead to the contamination of the Sample Blank
next sample to be injected. The carryover will affect the 1 4448995 13825 0.31 Accepted
accuracy of the quantification of the next sample. The 2 4451716 9014 0.20 Accepted
carryover assay was carried out by injecting a blank 3 4462653 11112 0.25 Accepted
(methanol) after caffeine standard solution (0.2 mg-mL-1 in Mean 4450355 11419 0.26 Accepted
methanol). The level of carryover was determined by RSD 0.04 29.8

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21
3. Detector Table 8: Summarized results for detector linearity test
3.1 Wavelength accuracy Conc. Peak Peak Peak Mean
RSD
Wavelength accuracy is defined as the deviation of the (mg/ml) area1 area2 area3 area
wavelength reading at an absorption or emission band from 0.05 1098062 1098412 1094059 1096844 0.22
the known wavelength of the band. The accuracy and 0.1 2112728 2130427 2129612 2124256 0.47
sensitivity of the measurement will be compromised if 0.2 4382290 4396149 4394306 4390915 0.17
there is a wavelength accuracy problem. Fig 4 shows the 0.4 8504402 8552800 8524150 8527117 0.29
UV spectrum for caffeine solution in the range of 190 to 1 23612372 23651803 23713554 23659243 0.22
380nm. The obtained λmax and λmin of this spectrum prove Correlation
0.9992
coefficient, r =
that the detector has no problem regarding to wavelength
Accept/Not Accept Accepted
accuracy.

Fig 5: Graph for Detector linearity

4. Column Oven
Capacity factor, k’ of an analyte decreases with as
temperature increases, and hence the retention of the
analysis decreases with temperature [16, 17]. The ability to
maintain an accurate column temperature is highly essential
to achieve the desired retention time and resolution
Fig 4: UV spectrum for caffeine
requirements in the separation process.
3.2 Detector response Linearity
The temperature accuracy of the column oven is evaluated
The detector linearity is very important when the purpose
by placing a calibrated thermometer in the column
of work is to carry out quantitative analysis. The linearity
compartment to measure the actual compartment
of the detector is important to the accuracy for the peak
temperature. As table 9 shows, the column oven is not
area and peak height comparison between standards and
accurate for temperature more than 50oC and hence it is
samples and accordingly to the determination of analyte (s)
highly recommended not to use this HPLC for
in these samples. Results for detector linearity test have
chromatographic conditions which require column
been summarized and graphed in Table 8 and figure 5
temperature more than 50oC.
respectively.

Table 9: Results for performance verification of column oven

Set Temperature (oC) Reading (oC) Correction factor (oC) Experimental Temperature (oC)
30 28.2 0.0 28.2
40 38.1 +0.1 38.2
50 48.3 +0.2 48.5
60 57.5 -0.4 57.1
80 75.2 -0.5 74.7
Conclusion And hence, it is quite logical to use this HPLC instrument
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