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SOP for Calibration of Shimadzu HPLC (Prominence – i LC – 2030)

OBJECTIVE 
To lay down a procedure for the Calibration of the HPLC Shimadzu, (Prominence– i LC – 2030). 

SCOPE 
This procedure applies to the, HPLC Shimadzu (Prominence– i LC – 2030) located in the Quality Control Laboratory.
RESPONSIBILITY 
Execution : Chemist and above – QC department. 
Checking : Asst. Manager and above – QC department 

FREQUENCY 
Calibration shall be done every six months and after maintenance or replacement of spare parts. 


CALIBRATION PROCEDURE 

1. Calibration of the pump:

A. Flow rate Accuracy 
  • Connect the inlet and outlet tubing’s with a union. 
  • Purge the line with water and ensure that the flow line is free from air bubbles. 
  • Set the flow rate at 0.5ml / min, collect the mobile phase (water) in a dry 10mL volumetric flask and fill the volumetric flask up to the mark, note down the time. 
  • Calculate the flow rate by using the formula (Volume of water/ Time in sec) x 60 
  • Repeat the above procedure for 1.0ml/min, 2.0ml/min, 3.0ml/min. Record the results in Attachment-I. 
  • Acceptance Criteria: ± 2% of the flow rate 
B. Flow rate Consistency. 
  • Record the RSD of the retention time of the uracil or caffeine peak under the test injector repeatability and record the results in Attachment-I. 
  • Acceptance Criteria: % RSD should not be more than 1% 
C. For gradient valve 
  • Install a union in place of the column & flush solvent lines (A) at a flow rate of 2ml/min with water. 
  • Fill reservoir A with 100% HPLC grade water and reservoir B, C & D with 0.3% acetone in water as mobile phase. 
  • Set the following time program. 
  • Use the detector at a wavelength of 254 nm. 
  • Record the value of the gradient valve test in Attachment-I and repeat the procedure for C and D line. 


2. Calibration of the Injector:

A. Volume Accuracy 
  • Take HPLC grade water in one of the vial and stoppered with septa. 
  • Weigh the vial with water (W1). 
  • Place the vial in the sample compartment. Set the system to inject 10 injections with 10µl- injections volume. 
  • Take the vial and reweigh it (W2). 
  • Difference in weight gives the weight of water drawn by the auto injector. (W1-W2). 
  • Calculate the quantity with drawn by the auto injector per injection. 
  • Calculate the volume with drawn by the auto injector per injection by the formulae (W1-W2)/10/ 0.99602, Where 0.99602 is density of water at 25°C. 
  • The volume of water drawn by the auto injector per injection should be within ± 0.5 % of the set value. 
  • Repeat the procedure with 20µl and 50µl. 

B. Repeatability of the Results 

Chromatographic condition 
   Column : BDS Hypersil or equivalent 250 x 4.6 mm, 5.0µm 
   Flow rate : 1.0 ml/min 
   Injector Volume: 20µl 
   Wavelength : 254 nm 
   Mobile phase : Methanol 

Standard preparation (20ppm): 
  • Transfer about 25 mg of Uracil or caffeine to a 100ml volumetric flask, add 70ml of Methanol, sonicate to dissolve and make up the volume with Methanol. Dilute 4 ml of resulting solution to 50ml with mobile phase. 
  • Inject the standard preparation five times and record the observed area in Attachment-I. 
Acceptance Criteria: % RSD of the area of Uracil or caffeine should not be more than 1%. 

C. Injector Linearity 
  • For chromatographic condition and standard preparation refer section B. 
  • Inject 10, 20, 30, 40 & 50µl of the standard preparation in duplicate. Calculate the average area counts corresponding to each set of injection. 
  • Plot a graph of mean area against injection volume and calculate the correlation coefficient. 
Acceptance Criteria: Correlation factor - NLT 0.999.
 
3. Calibration of the Detector:

A.  Detector Linearity

For chromatographic condition refer section B (Repeatability of the Results). 
Standard stock solution (250ppm): 
  • Transfer about 25 mg of Uracil or caffeine to a 100ml volumetric flask, add 70ml of Methanol, sonicate to dissolve and make up the volume with Methanol. 
  • Take 2ml, 4ml, 6ml, 8ml and 10ml of stock solution respectively in 50 ml of volumetric flask, and dilute up to the mark with methanol. 
  • Inject all the solution in duplicate and record the peak response in Attachment-I. 
  • Plot the graph of concentration against mean area of Uracil or caffeine and calculate the correlation factor. 
Acceptance Criteria: Correlation factor - NLT 0.999 

4. Calibration of the Column Oven:
  • Set the column oven temperature to 30°C. 
  • Allow the system to attain set temperatures. 
  • Read the temperatures using data logger. 
  • Repeat the same for oven temperature 50°C. 
Acceptable temperature range for column oven is + 2°C. 

ATTACHMENTS 
Attachments 1: Calibration Record of HPLC. 
An Image/Link below is provided (as is) to download Annexure
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ABBREVIATIONS 
UV – Ultra Violet 
HPLC – High Performance Liquid Chromatography 
QC – Quality Control 

REVISION HISTORY 
Nil 

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