Available online at http://www.urpjournals.com
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
Good analytical results are essential in order to take reliable
decisions. Analytical measurements affect the daily lives of
every citizen. Sound, accurate and reliable analytical
measurements are fundamental to the functioning of
modern society. A wrong result can have an enormous
social and economic impact [1]. The correctness of
measurements and measuring instruments is one of the key
prerequisites to ensure the quality of products and services,
and the accuracy of the instruments must be consistent with
their intended use [2].
Calibration and verification are the most important actions
to ensure the correct indication of measuring instruments
[2]. Regular calibration of measuring instruments should be
carried out in agreement with the implemented quality
systems. The industrial metrology ensures the appropriate
functioning of measurement instruments used in industry as
well in production and testing processes, in order to
guarantee the quality of life for citizens and for academic
research [3].
Verification is the confirmation, based on evidences (facts,
test results) that some specified requirement has been
fulfilled. The result from a verification assay will show if
the measuring equipment is in agreement with its required
specifications, which are generally expressed as tolerances
[4]. The verification of measuring instruments includes
testing and requires the availability of clear specifications
and acceptance/refusal criteria [5]. Verification provides
means of checking that the deviations between the values
displayed by a measuring instrument and the corresponding
18
known values of measured quantity are under control.
High-Performance Liquid Chromatography (HPLC) is one
of the foremost analytical techniques widely used in
analytical laboratories for the analysis of pharmaceuticals
and chemicals [6-8], foods [9 -11], cosmetics samples and
so on [12, 13]. In order to provide a high level of assurance
that the data generated from the HPLC analysis are reliable,
the performance of the HPLC system should be monitored
at regular intervals.
The performance of an HPLC system can be evaluated by
examining the key functions of the various modules that
comprise the system. The common HPLC performance
attributes and the acceptance/refusal criteria are presented
in Table 1. The acceptance/refusal criteria values for these
attributes are based on the values suggested by Herman
Lam in the chapter “Performance Verification of the
HPLC” of his book [14].
2. Experimental
2.1 Instruments and apparatuses
A low pressure quaternary HPLC instrument comprises
LC-10ATvp pump, SIL-10ADvp auto sampler, SPD10AVvp UV-Vis detector and CTO-10AVP column oven
(Shimadzu, Japan) has been verified for its proper
functioning. Analytical balance (METTLER Toledo,
Switzerland), Calibrated stop watch (Treaceble®
stopwatch,
JUMBO DIGIT, VWR,
China) and
thermometer (Service Testo, Germany) have been used.
2.2 Materials and chemicals
ODS analytical column (150 X 4.6 mm, 5μm, Waters,
USA), Class A 25mL volumetric flasks (Pyrex, Germany),
International Journal of Chromatographic Science 2013, 3(1): 18-23
methanol HPLC grade (BDH, USA), Acetonitrile HPLC
grade (J.T. Baker, USA) and caffeine (Fluka, SigmaAldrich, China, purity ≥ 0.99%) were employed for this
work.
Table 1: Performance attributes and their acceptance/
refusal criteria for HPLC verification
Performance
Acceptance/refusal
Module
Attributes
criteria
± 1% of the set
Pump
Flow rate accuracy
flow rate
± 1% of the step
Gradient accuracy
gradient
composition
Auto injector
Precision
RSD ≤ 1%
Linearity
r ≥ 0.999
Carryover
< 1%
Wave length
Detector
± 2nm
accuracy
Linearity of
r ≥ 0.999
response
Column
Temperature
± 2 oC of the set
oven
accuracy
temperature
2.3 Methods
2.3.1 Chromatographic condition (For Auto sampler
and Detector verification)
A mobile phase containing 85% water and 15% acetonitrile
was pumped through the analytical column (ODS, 150 X
4.6mm, 5μm) at a flow rate of 2 mL/minute. The test
solutions, caffeine in methanol, were injected and detected
at 272nm. The column temperature was ambient.
2.3.2 Verification Test for different modules of HPLC
2.3.2.1 Pump Module:
Flow Rate Accuracy
The flow-rate of the pump was set at 2 mL/min. and the
time required to fill a 25 mL volumetric flask was
measured using the calibrated stopwatch.
Gradient Accuracy and Linearity
The accuracy and linearity of the gradient solvent delivery
has been verified indirectly by monitoring the absorbance
change as the binary composition of the two solvents
changes from two different channels. The LC gradient has
four channels: A, B, C and D. The test was performed for
two channels at a time. Channel A is filled with a pure
solvent, methanol, while channel B is filled with a solvent
containing a UV-active tracer, caffeine. The gradient
profile is programmed to pump from solvent A only for two
minutes and then to decrease the composition of Solvent A
by 10% and to increase solvent B by the same percent and
allowing to pump for two minutes at each change. The
absorbance change at each composition change is measured
and expressed as height H in the plot of absorbance versus
solvent composition. The linearity of the gradient delivery
was verified by plotting the absorbance at various mobile
phase compositions versus the theoretical composition. The
entire process was repeated for channels C and D.
2.3.2.2 Injector Module:
Precision
The precision of the injector was demonstrated by making
six replicate injections from a sample (caffeine 0.05 mg/mL
19
in methanol). The relative standard deviation (RSD) of the
response of the injections was calculated to evaluate the
precision.
Linearity
The linearity of the injector was demonstrated by making
injections of 5, 10, 20, 30 and 50 μL of the caffeine
solution (0.05 mg/mL in methanol). The response of the
injections at each injection volume was plotted against the
injection volume. The correlation coefficient of the plot
was used in the evaluation of the injection linearity.
Carryover
Carryover was evaluated by injecting a blank (methanol)
after a sample that contains a high concentration of analyte
((0.2 mg/mL of caffeine in methanol). The response of the
analyte found in the blank sample expressed as a
percentage of the response of the concentrated sample was
used to determine the level of carryover.
2.3.2.3. UV-Visible Detector Module:
Wavelength Accuracy
The wavelength verification of the UV range was
performed by filling a flow cell with a
Caffeine solution (0.015mg/mL in methanol) and the UV
spectrum was collected in the range of 200 to 400nm. The
obtained scan should have λmax at about 272nm and λmin at
about 244nm.
Linearity of Response
The linearity of the detector response was checked by
injecting a series of standard solutions of caffeine (0.05,
0.1, 0.2, 0.4 and 1 mg/mL) to the chromatographic system.
From the plot of response versus the concentration of the
solutions, the correlation coefficient between sample
concentration and response was calculated to determine the
linearity.
2.3.2.4. Column Oven module:
The accuracy of the oven was checked at 30, 40, 50, 60 and
70 oC using the calibrated thermometer. The correction
factors obtained from the calibration certificate of the
thermometer were considered to get the experimental
temperature.
Results and Discussion
1. Pump module
1.1. Flow rate Accuracy
One of the key performance requirements for the pump
module is the ability to maintain accurate and consistent
flow of the mobile phase, which will be necessary to
provide stable and repeatable interactions between the
analytes and the stationary phase [15]. Poor flow-rate
accuracy will affect the retention time of the separation. As
shown in table 2, the results for flow rate accuracy for all of
the four suction channels is in the range of 99.04 to 100.4
% and this good result indicates that the pump performance
is superior.
1.2. Gradient Accuracy and Linearity
For gradient accuracy, the ability of the pump to deliver the
mobile phase at different solvent strengths over the time by
varying the composition of the mobile phase accurately is
fundamental to get the adequate chromatographic
separation and reproducibility.
As shown in fig 1, the absorbance increased step by step as
the composition of the caffeine increased. Similarly, the
International Journal of Chromatographic Science 2013, 3(1): 18-23
absorbance decreased to zero as the composition of the
caffeine decreased to zero. The height increased due to the
change in caffeine composition was measured by the LC
solution software from the baseline and these heights have
been used for the determination of gradient accuracy and
gradient linearity. The gradient linearity is determined by
graphing the percentage composition of caffeine versus the
respective obtained height. As shown in figure 2, the
relationship between percentage of the composition of
caffeine and the obtained height is linear with a correlation
coefficient of 0.9998.
Table 3 and Table 4, shows the
result for gradient accuracy for channel A and B and
channel C and D respectively. The accuracy is calculated
by determining the response factor for each percentage
change and comparing it with the average response factor.
The obtained accuracies at each level are in agreement with
the acceptance/refusal criteria for all channels.
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
Rep
Vol.
(mL)
Theoretical
time (min)
1
2
3
4
5
25
25
25
25
25
12.5
12.5
12.5
12.5
12.5
a
Time
(min:Sec
)
12:26
12:25
12:26
12:27
12:26
b
Time
(min)
12.43
12.42
12.43
12.45
12.43
Line B
Accu
racy
(%)
99.44
99.36
99.44
99.60
99.44
a
b
12:30
12;29
12:28
12:27
12:23
12.5
12.48
12.47
12.45
12.38
Time
(min:Sec)
Line C
Accur
acy
(%)
100.00
99.84
99.76
99.60
99.04
Time
(min)
Table 3: Gradient accuracy results for channel A and B.
Channel
Channel
Time
Height 1
Height 2
A (%)
B (%)
(min)
(Abs Units)
(Abs Units)
100
0
2
0
0
90
10
4
69506
70058
80
20
6
145847
144592
70
30
8
221595
218145
60
40
10
292574
292520
50
50
12
368022
370379
40
60
14
438919
438899
30
70
16
512502
514026
20
80
18
583598
584747
10
90
20
661655
660386
0
100
22
733540
730582
a
a
b
12:33
12:30
12:29
12:27
12:28
12.55
12.5
12.48
12.45
12.47
Time
(min:Sec)
Height 3
(Abs Units)
0
70356
149227
220563
293820
368457
438377
513741
591672
659681
731038
Time
(min)
Line D
Accur
acy
(%)
100.40
100.00
99.84
99.60
99.76
a
b
12:28
12:26
12:27
12:27
12:27
12.47
12.43
12.45
12.45
12.45
Time
(min:Sec)
Average
(Abs Units)
0
69973
146555
220101
292971
368953
438732
513423
586672
660574
731720
Mean
a
Ave. Height
(Abs Units)
0
71883
147586
218975
297156
367503
440618
515488
584921
661889
730515
Mean
a
Accur
acy
(%)
99.76
99.44
99.60
99.60
99.60
Time
(min)
Response
Factor
NA
6997.3
7327.8
7336.7
7324.3
7379.1
7312.2
7334.6
7333.4
7339.7
7317.2
7300.2
b
Acc.
(%)
98.6
100.4
100.5
100.3
101.1
100.2
100.5
100.5
100.5
100.2
100.3
Response factor = Average height/percentage composition of caffeine
Accuracy (%) = Response factor* 100/Average response factor
b
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
Channel C Channel
Time
Height 1
Height 2
Height 2
(%)
B (%)
(min)
(Abs Units)
(Abs Units)
(Abs Units)
100
0
2
0
0
0
90
10
4
70567
72446
72635
80
20
6
143868
148397
150494
70
30
8
216505
219043
221376
60
40
10
292260
295224
303983
50
50
12
363730
368817
369963
40
60
14
437677
441136
443041
30
70
16
511447
514552
520466
20
80
18
581062
586289
587411
10
90
20
659430
661308
664928
0
100
22
729972
730282
731290
2. Auto injector module
2.1. 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
20
Response
Factor
NA
7188.3
7379.3
7299.2
7428.9
7350.1
7343.6
7364.1
7311.5
7354.3
7305.1
7332.4
b
Acc.
(%)
98.7
100.6
99.5
101.3
100.2
100.2
100.4
99.7
100.3
99.6
100.1
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. No meaningful
comparison between the responses of the sample and the
International Journal of Chromatographic Science 2013, 3(1): 18-23
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. Table 5 shows the result
for the auto sampler precision test and it proves that the
auto sampler is precise as the relative standard deviation for
the six injections is less than 1%.
Fig 1: Chromatogram for gradient accuracy test of channel
A and B
Fig 2: Graph which shows the relationship between
percentage composition of caffeine and height for channel
A and B
Table 5: Result for the auto sampler precision
Replicate
Peak area
1
1098235
2
1101507
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
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.
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
correlation coefficient is 0.999998 which is more than the
acceptance/refusal criteria, 0.999.
Table 6: Results for Auto injector linearity with its acceptance/refusal criteria
Inj. vol (µL)
Peak Area 1
5
533967
10
1063166
20
2105973
50
5269317
Correlation coefficient, r
Accepted/Not Accepted
Peak area 2
531499
1064724
2113965
5282999
Fig 3: Graph for the relationship between volume of
injection and peak area
2.3. Carry over
Small amounts of analyte may get carried over from the
sample injected before and lead to the contamination of the
next sample to be injected. The carryover will affect the
accuracy of the quantification of the next sample. The
carryover assay was carried out by injecting a blank
(methanol) after caffeine standard solution (0.2 mg-mL-1 in
methanol). The level of carryover was determined by
21
Peak area 3
533654
1067951
2114930
5306851
Average
533040
1065280
2111623
5286389
RSD
0.252081
0.229097
0.232830
0.359324
0.999998
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
use it for quantitative analysis.
Table 7: Carry over test results for the auto injector
Replicate
1
2
3
Mean
RSD
Peak Area
Sample
4448995
4451716
4462653
4450355
0.04
Blank
13825
9014
11112
11419
29.8
Carry
over (%)
Accepted/Not
Accepted
0.31
0.20
0.25
0.26
Accepted
Accepted
Accepted
Accepted
International Journal of Chromatographic Science 2013, 3(1): 18-23
3. Detector
3.1 Wavelength accuracy
Wavelength accuracy is defined as the deviation of the
wavelength reading at an absorption or emission band from
the known wavelength of the band. The accuracy and
sensitivity of the measurement will be compromised if
there is a wavelength accuracy problem. Fig 4 shows the
UV spectrum for caffeine solution in the range of 190 to
380nm. The obtained λmax and λmin of this spectrum prove
that the detector has no problem regarding to wavelength
accuracy.
Table 8: Summarized results for detector linearity test
Conc.
Peak
(mg/ml)
area1
0.05
1098062
0.1
2112728
0.2
4382290
0.4
8504402
1
23612372
Correlation
coefficient, r =
Accept/Not Accept
Peak
area2
1098412
2130427
4396149
8552800
23651803
Peak
area3
1094059
2129612
4394306
8524150
23713554
Mean
area
1096844
2124256
4390915
8527117
23659243
RSD
0.22
0.47
0.17
0.29
0.22
0.9992
Accepted
Fig 5: Graph for Detector linearity
Fig 4: UV spectrum for caffeine
3.2 Detector response Linearity
The detector linearity is very important when the purpose
of work is to carry out quantitative analysis. The linearity
of the detector is important to the accuracy for the peak
area and peak height comparison between standards and
samples and accordingly to the determination of analyte (s)
in these samples. Results for detector linearity test have
been summarized and graphed in Table 8 and figure 5
respectively.
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
requirements in the separation process.
The temperature accuracy of the column oven is evaluated
by placing a calibrated thermometer in the column
compartment to measure the actual compartment
temperature. As table 9 shows, the column oven is not
accurate for temperature more than 50oC and hence it is
highly recommended not to use this HPLC for
chromatographic conditions which require column
temperature more than 50oC.
Table 9: Results for performance verification of column oven
Set Temperature (oC)
Reading (oC)
Correction factor (oC)
30
28.2
0.0
40
38.1
+0.1
50
48.3
+0.2
60
57.5
-0.4
80
75.2
-0.5
Conclusion
The performance of an HPLC system can be evaluated by
examining the key functions of the various modules that
comprise the system. The common HPLC performance
attributes that must be qualified are pump, auto injector,
column oven and detector. The obtained verification results
for all of the attributes are complying with the
acceptance/refusal criteria values as per Herman Lam
suggestion. However, the column oven is out of the
acceptance/refusal criteria for temperature more than 50 oC.
22
Experimental Temperature (oC)
28.2
38.2
48.5
57.1
74.7
And hence, it is quite logical to use this HPLC instrument
for the day to day analytical purpose
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Source of support: Nil; Conflict of interest: None declared
23
International Journal of Chromatographic Science 2013, 3(1): 18-23