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

OBJECTIVE 

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

SCOPE 

This procedure applies to the HPLC Shimadzu (Prominence– i LC – 2030) 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 after spare parts' maintenance or replacement. 

ALSO READ: SOP for Operation of Shimadzu HPLC (Prominence – i LC – 2030)

CALIBRATION PROCEDURE 

1. Calibration of the pump:

A. Flow rate Accuracy 

• Connect the inlet and outlet tubing 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 a sec) x 60 
• Repeat the above procedure for 1.0ml/min, 2.0ml/min, and 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 reservoirs 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 lines. 

ALSO READ: SOP for Shimadzu LabSolutions

2. Calibration of the Injector:

A. Volume Accuracy 

• Take HPLC-grade water in one of the vials and stopper with septa. 
• Weigh the vial with water (W1). 
• Place the vial in the sample compartment. Set the system to inject 10 injections with a 10µl- injection volume. 
• Take the vial and reweigh it (W2). 
• The difference in weight gives the weight of water drawn by the auto-injector. (W1-W2). 
• Calculate the quantity withdrawn by the auto-injector per injection. 
• Calculate the volume withdrawn by the auto-injector per injection by the formulae (W1-W2)/10/ 0.99602, Where 0.99602 is the density of water at 25°C. 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 the resulting solution to 50 ml with the 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 conditions and standard preparation refer to section B. 
• Inject 10, 20, 30, 40 & 50µl of the standard preparation in duplicate. Calculate the average area counts corresponding to each set of injections. 
• Plot a graph of the 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 conditions refer to 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 the volumetric flask, and dilute up to the mark with methanol. 
• Inject all the solutions in duplicate and record the peak response in Attachment-I. 
• Plot the graph of concentration against the mean area of Uracil or caffeine and calculate the correlation factor. 

Acceptance Criteria: Correlation factor - NLT 0.999

ALSO READ: Troubleshooting Problems with Retention Variation

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 the data logger. 
• Repeat the same for oven temperature 50°C. 

The acceptable temperature range for the column oven is + 2°C.

ATTACHMENTS 

Attachments 1: Calibration Record of HPLC.

ABBREVIATIONS 

UV – Ultra Violet 

HPLC – High-Performance Liquid Chromatography 

QC – Quality Control 

REVISION HISTORY 

Nil

ALSO READ: SOP for Operation of HPLC (Shimadzu LC-2030C 3D (PDA))