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HPLC Calibration Parameters
Following Parameters are used during calibration of
HPLC:
·
Flow rate accuracy
test
·
Retention time
reproducibility:
·
Gradient composition:
·
Calibration of
injector:
·
Injection
precision:
·
Injector & detector linearity:
·
Carry over test
·
Injection
accuracy:
·
Wave length accuracy:
·
Temperature
accuracy:
·
Sampler
cooler temperature accuracy
·
Calibration of pda detector (spd – m20a):
·
Lamp energy
CALIBRATION of HPLC System
(SHIMADZU LC2030C- PROMINACE -i)/(PDA-Detector)
(1)
FLOW RATE ACCURACY TEST:
Mobile Phase: water
Stop Watch ID. No: …………………….
Stop Watch Calibration Due on:
……………………….
Sr. No
|
Set
flow rate
|
Time
in minutes
|
Observed vol. at 250C
=
{(Wt.
of V. flask +water) –(Wt. of V. flask)}/0.997
|
Observed flow in ml =
Observed Vol.
Observed time
|
Limit
( ±1%)
|
Remarks.
|
1
|
0.5 ml/min
|
20 minute
Observed Time =
|
±
0.005
|
Complies/
Does not comply
|
||
2
|
1.0
ml/min
|
10
minute
Observed Time =
|
± 0.01
|
Complies/
Does
not comply
|
||
3
|
2.0
ml/min
|
5
minute
Observed Time =
|
± 0.02
|
Complies/
Does
not comply
|
||
4
|
2.5
ml/min
|
4
minute
Observed Time =
|
± 0.025
|
Complies/
Does
not comply
|
Remarks: Pass/ Fail
(2)
RETENTION TIME REPRODUCIBILITY:
Caffeine
Batch No. Make: Used before
date:
Column ID:
Chromatographic
conditions:
HPLC
Condition : Column C18
150 x 4.6 mm, 5µ.
Detector : 272 nm (for caffeine)
Injection
volume : 20 µl.
Mobile phase : Methanol: water (70:30)
Flow
rate : 1.0 ml/min.
Take ………………..mg of Caffeine and dilute to 100 ml with mobile phase.
Further dilute 2 ml to 50 ml with mobile phase.
Injection No.
|
Retention time
|
Limit
|
1
|
% RSD not more than 1.0
|
|
2
|
||
3
|
||
4
|
||
5
|
||
6
|
||
Mean
|
||
SD
|
||
% RSD
|
Remarks: Pass /Fail
(3) GRADIENT COMPOSITION:
Composition
|
Composition Using A & C
|
Composition Using B & D
|
Limit
|
||
m.v
|
% of composition
|
m.v
|
% of composition
|
||
C100
|
|||||
C90
|
89.0 to
91.0
|
||||
C50
|
49.0 to
51.0
|
||||
C10
|
9.0 to
11.0
|
||||
Calculation: for channel A & C
Actual concentration of 10 %
(C) = ---------------- x100
Actual concentration of 50 %
(C) = ---------------- x100
Actual concentration of 90 %
(C) = ---------------- x100
Calculation: for channel B & D
Actual concentration of 10 %
(D) = ---------------- x100
Actual concentration of 50 %
(D) = ---------------- x100
Actual concentration of 90 %
(D) = ---------------- x100
Remarks: Pass /Fail
(4) Calibration of
Injector:
(A). INJECTION PRECISION:
Injection No.
|
Area
|
Limit
|
1
|
% RSD not more than 2.0
|
|
2
|
||
3
|
||
4
|
||
5
|
||
6
|
||
Mean
|
||
SD
|
||
% RSD
|
Remarks: Pass/ Fail
(B) INJECTOR & DETECTOR LINEARITY:
Injection No.
|
Injection Volume
|
Area
|
Limit
|
1
|
5
|
||
2
|
10
|
||
3
|
20
|
||
4
|
30
|
||
5
|
40
|
||
6
|
50
|
||
7
|
100
|
||
Correlation coefficient
|
NLT 0.999
|
||
Remarks: Pass/ Fail
(C) CARRY OVER TEST: (Use
250 mcg/ml caffeine standard solution and mobile phase as a
blank)
Sr. No.
|
Mean area of solution
|
Area of caffeine in blank injection
|
% Carry Over
|
Limit
|
NMT 0.05%
|
Remarks: Pass /Fail
(D) INJECTION ACCURACY:
Sr. No
|
Weight of vial + water
( W1
in mg )
|
Set volume
µl
|
Number of
injections
‘n’
|
Wt. of
vial + water in mg (W2)
( after 6 injections )
|
Avg. amount of injected water in µl
|
Acceptance
value
in µl
|
20
|
6
|
19.6 to
20.4 µl
|
Remarks: Pass /Fail
Calculation:
W1- W2
Average Volume of
Injection = ------------------
0.997 X
n
(5) WAVE LENGTH ACCURACY:
Column ID:
_______________________
(i) From wave length Maxima (202 nm – 210
nm):
S. No.
|
Wavelength
|
Absorbance
|
Observed maxima at
|
Limit
|
1.
|
202
|
205 ± 2 nm
|
||
2.
|
203
|
|||
3.
|
204
|
|||
4.
|
205
|
|||
5.
|
206
|
|||
6.
|
207
|
|||
7.
|
208
|
|||
8.
|
209
|
|||
9.
|
210
|
Remarks: Pass /Fail
(ii) From wave length Minima (240 nm –
248 nm):
S. No.
|
Wavelength
|
Absorbance
|
Observed Minima at
|
Limit
|
1.
|
240
|
245 ± 2 nm
|
||
2.
|
241
|
|||
3.
|
242
|
|||
4.
|
243
|
|||
5.
|
244
|
|||
6.
|
245
|
|||
7.
|
246
|
|||
8.
|
247
|
|||
9.
|
248
|
Remarks: Pass /Fail
(iii) From wave length (268 nm – 276 nm):
S. No.
|
Wavelength
|
Absorbance
|
Observed maxima at
|
Limit
|
1.
|
268
|
273 ± 2 nm
|
||
2.
|
269
|
|||
3.
|
270
|
|||
4.
|
271
|
|||
5.
|
272
|
|||
6.
|
273
|
|||
7.
|
274
|
|||
8.
|
275
|
|||
9.
|
276
|
Remarks: Pass /Fail
(6) Temperature Accuracy:
Oven Temperature:
Thermometer ID:
_________________ Calibration
certificate No.: ____________
Calibration Valid up to:
_____________
Sr. No
|
Set
Temperature
|
Displayed Temp.
|
Observed temp of thermometer
|
Limits:
|
1
|
25 ° C
|
24 to 26
° C
|
||
2
|
40 ° C
|
39 to 41
° C
|
||
3
|
60 o
C
|
59 to 61
o C
|
Remarks: Pass /Fail
(7) Sampler cooler temperature accuracy:
Thermometer ID No. ……………………………. Calibration due date
……………..……….
Sr. No
|
Set
Temperature
|
Displayed Temp.
|
Observed temp of thermometer
|
Limits
|
1
|
4o C
|
3°C to 5°C
|
||
2
|
10° C
|
9°C to 11°C
|
||
3
|
40 ° C
|
39°C to 41°C
|
Remarks: Pass /Fail
(8) CALIBRATION OF PDA DETECTOR (SPD – M20A):
(A) Detector performance:
PDA Detector Precision:
Preparation of Caffeine stock solution (200
mcg):
Weigh accurately .......... mg Caffeine in 100 ml volumetric flask; add
60 ml mobile phase to dissolve and make up the volume with same.
Preparation of Caffeine solution (20 mcg):
Transfer .............. ml of
Caffeine stock solution in 50 ml volumetric flask and make up and mix with mobile phase.
Solution Used:
Injection
No.
|
Retention
Time
|
Area
Response
|
Observed
% RSD
|
Acceptable
% RSD
|
Test Result
|
1.
|
NMT 1.0 % For
Retention Time
|
Pass/fail
|
|||
2.
|
|||||
3.
|
|||||
4.
|
NMT 2.0 % For
Area
|
||||
5.
|
|||||
6.
|
|||||
Avg.
|
|||||
SD
|
|||||
RSD
|
|||||
Remarks: Pass /Fail
(B) PDA Detector Linearity:
Preparation
of Caffeine stock solution (200 mcg):
Weigh accurately ................ mg of Caffeine in 100 volumetric
flasks.
Preparation
of Caffeine stock solution (10 mcg):
Transfer .............. ml of Caffeine Stock solution in 100 ml
volumetric flask.
Preparation
of Caffeine stock solution (20 mcg):
Transfer .............. ml of Caffeine Stock solution in 50 ml volumetric
flask.
Preparation
of Caffeine stock solution (40 mcg):
Transfer .............. ml of Caffeine Stock solution in 10 ml volumetric
flask.
Preparation
of Caffeine stock solution (80 mcg):
Transfer .............. ml of Caffeine Stock solution in 25 ml volumetric
flask.
Preparation
of Caffeine stock solution (100 mcg):
Transfer .............. ml of Caffeine Stock solution in 10 ml volumetric
flask.
S. No.
|
Concentration of Caffeine
|
Area Response
|
Observed
( r )
|
Acceptable
( r )
|
Test
Results
|
1.
|
10 mcg
|
NLT 0.999
|
Pass / Fail
|
||
2.
|
20 mcg
|
||||
3.
|
40 mcg
|
||||
4.
|
80 mcg
|
||||
5.
|
100 mcg
|
Remarks: Pass /Fail
(9) LAMP ENERGY:
Observation
|
Limit: NLT 400
|
Remarks:
Pass /Fail
Conclusion: Instrument
meets / does not meet the acceptance criteria.
