Document Title : HPLC CALIBRATION

Reference SOP : PROCEDURE FOR OPERATION&CALIBRATION OF HPLC

Reference SOP No. : PB/SOP/QCD/001/01 Format No. : F/PB/QCD/001/01
Annexure No. : III Page No. : 1 of 1




HPLC ID No: ------------------------------- Column ID No : -------------------------
Make : ------------------------------ Model : --------------------------
Date of calibration: ------------------------ Calibration due Date: ---------------------------
Frequency : Half Yearly
1. FLOW RATE:
Weight per ml of water
Wt of Empty Pycnometer g
Wt / ml

=
g/ml
Wt of Filled Pycnometer g
Wt of Water g 25.0



Set flow
Wt. of
empty
Cylender
(W1)
Wt. of
Cylender +
Water after
10 minutes
(W2)
Flow rate
(ml/min)=
[(W2-W1)/10 x
wt/ml]
Average
flow Rate
(ml/min)
Limit + 2.0%
Minimum
(ml/min)
Maximu
m
(ml/min)
1.0 ml/min
g g
0.98 1.02 g g
g g
2.0 ml/min g g 1.96 2.04
Document Title : HPLC CALIBRATION
Reference SOP : PROCEDURE FOR OPERATION&CALIBRATION OF HPLC
Reference SOP No. : PB/SOP/QCD/001/01 Format No. : F/PB/QCD/001/01
Annexure No. : III Page No. : 2 of 1




2. Linearity (Detector) :
Reagent Name : --------------------------------------- Reagent B.No: -----------------------------
Mobile phase : 70:30Methanol&Water Balance ID.No:------------------------------
Column : ----------------------------------- Column ID .No:-----------------------------
Wave length : ---------------------------------- Flow Rate (1ml/Min) :---------------------

Sample Solution A : Weigh accurately about ____(100 mg) of Caffeine Anhydrous Pure in 100 ml of
Volumetric Flask, dissolve and make up to mark with Mobile Phase.

Sample Solution B: dilute 1 ml of this solution A to 100 ml with Mobile Phase (10 ppm).

Sample Solution C: dilute 2 ml of this solution B to 100 ml with Mobile Phase (20 ppm).

Sample Solution D: dilute 3 ml of this solution C to 100 ml with Mobile Phase (30 ppm).

Sample Solution E: D dilute 4 ml of this solution D to 100 ml with Mobile Phase (40 ppm).





Document Title : HPLC CALIBRATION
Reference SOP : PROCEDURE FOR OPERATION&CALIBRATION OF HPLC
Reference SOP No. : PB/SOP/QCD/001/01 Format No. : F/PB/QCD/001/01
Annexure No. : III Page No. : 3 of 1






S.No
Concentrati
on
Area Mean Area
Correlation
Coefficient (r)
Acceptance
Criteria
01 10 ppm



0.98 to
1.000

02 20 ppm



03 30ppm



04 40ppm



05 50ppm




Calibrated by: ------------------- Checked by: --------------------
Date : ----------------- Date : -------------------


Document Title : HPLC CALIBRATION
Reference SOP : PROCEDURE FOR OPERATION&CALIBRATION OF HPLC
Reference SOP No. : PB/SOP/QCD/001/01 Format No. : F/PB/QCD/001/01
Annexure No. : III Page No. : 4 of 1




3. Precision (Detector)
Sample Solution (20 ppm) : Dilute 2.0 ml of Solution A to 100ml with Mobile Phase (20 ppm). 6Replicates ,Inj

Inj. No Area Retention Time
01
02
03
04
05
06
Average
STDEV
% of RSD


Calibrated by: ------------------- Checked by: --------------------
Date : ----------------- Date : -------------------



Document Title : HPLC CALIBRATION
Reference SOP : PROCEDURE FOR OPERATION&CALIBRATION OF HPLC
Reference SOP No. : PB/SOP/QCD/001/01 Format No. : F/PB/QCD/001/01
Annexure No. : III Page No. : 5 of 1




4. Wavelenght Accuracy:
Sample Solution A : Weigh accurately about ____(100 mg) of Caffeine Anhydrous Pure in 100 ml of
Volumetric Flask, dissolve and make up to mark with Mobile Phase.
Sample Solution B: Further dilute 2 ml of this solution to 100 ml with Mobile Phase (20 g/ml).
S.No Wavelength (nm) Area Maxima at Acceptance Criteria
1 270


273nm
2 271
3 272
4 273
5 274
6 275
7 276


Instrument Calibration meets the requirement /doesn`t meets the requirements
Calibrated by: ------------------- Checked by: --------------------
Date : ----------------- Date : -------------------


Document Title : HPLC CALIBRATION
Reference SOP : PROCEDURE FOR OPERATION&CALIBRATION OF HPLC
Reference SOP No. : PB/SOP/QCD/001/01 Format No. : F/PB/QCD/001/01
Annexure No. : III Page No. : 6 of 1