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Calibration & Troubleshooting of HPLC

Calibration & Troubleshooting of HPLC

Calibration:

Chromatographic condition:

Column:               250 x 4.6 mm C18
Flow rate:             1.0 ml/min
Mobile phase:       A filtered and homogeneous mixture of methanol and distilled water (60:40)
Wavelength :        250 to 270 nm.
Injection volume: 20 µl    

1-Preparation of Injection:

Objective: To check the precision of  response through the 0.015 mg/ml solution of caffeine in                      mobile phase.
Stock solution of caffeine 0.1 mg: Accurate weight and transferred 0.1 mg of caffeine in 100 ml volumetric flask.Dissolved in methanol,make up to mark and mixed well.Diluted 10 ml this solution to 100 ml with mobile phase.
0.1 mg caffeine -----> 100 ml methanol ------> 10 ml --------> 100 ml mobile phase 
Caffeine solution 0.015 mg: Diluted 15 ml of this stock solution to 100 ml with mobile phase and mixed well.

100 ml mobile phase ------> 15 ml -------> 100 ml mobile phase


Procedure of Precision or repeatibility: Made five consecutive injection of the above solution (0.015 mg/ml) of caffeine and record the chromatograms. Calculate the relative standard deviation (RSD) of area and retention time of caffeine.
                            For Example:

Injection
Area of Caffeine
Retention time of caffeine
1
5067521
3.57
2
5066464
3.55
3
5074049
3.55
4
5022697
3.55
5
5084794
3.56

Area RSD=0.47%
RT RSD=0.25%

Conclusion: The RSD of the area of five injection of Caffeine 0.015 mg/ml solution is less than 1% and the RSD of the retention time of five injection of Caffeine 0.015 mg/ml solution is less than 0.5% which indicates consistency of the performance of the system.

2-Wavelength Calibration of Detector: 

Inject 20 µl of 0.015 mg/ml solution of Caffeine, Each time setting the detector at following five different wavelength i.e 265nm, 270nm, 275nm, 280nm, and 285nm recorded the area of the responses due to Caffeine.
                       For Example:

Wavelength (In nm)
Response (Area under Caffeine peak)
265
4304706
270
4981469
275
5102630
280
4481961
285
3217273

Conclusion: The detector stands calibration with respect to wavelength as it shows maximum response at 275 nm witch is the wavelength of maximum absorption of Caffeine.

3-Linearity of response:

Objective: To evaluate the performance of the detector.
                  Dilute 10 ml of stock solution (0.1 mg/ml of Caffeine) to 100 ml with mobile phase and mixed well. (i.e 0.01 mg/ml solution of Caffeine)
Prepared a series of solution having flowing concentration by diluting the 0.01 mg/ml solution with mobile phase as follows.
     For Example:           

Solution No.
Concentration in mg
ml of 10 mg taken
Final dilution with mobile phase
1
2
2
10 ml
2
3
3
10 ml
3
4
4
10 ml
4
5
5
10 ml
5
6
6
10 ml
6
7
7
10 ml

Procedure: Injected above Six solution separately to obtain chromatograms and recorded the area of Caffeine responses.
                 For Example:              

Solution No.
Concentration in mg
Response (Area under Caffeine peak)
1
2
640478
2
3
954535
3
4
1289530
4
5
1582273
5
6
1933292
6
7
2258708
  
 Plotted the graph of area responses against concentration.
Conclusion: The area responses produced by the Caffeine solution are found to be linear with correlation coefficient 0.99 indicate the satisfactory performance of the system.

4-Accuracy of flow rate:

Objective: To check the performance of pump.
Procedure: Set the flow rate to 0.5 ml, 1.0 ml, 1.5 ml, and 2.0 ml, respectively and record the volumes of solvent collected in 10 min.
Observation: The actual volumes of the solvent collected should be within the tolerance limits specified.
             For Example: 

Flow rate set at 10 min.
Volume collected per 10 min.
Tolerance
0.5 ml/min
5.03 ml
4.8 ml – 5.2 ml
1.0 ml/min
10.08 ml
9.8 ml – 10.2 ml
1.5 ml/min
15.10 ml
14.8 ml – 15.2 ml
2.0 ml/min
20.12 ml
19.8 ml – 20.2 ml

Conclusion: Pump is working satisfactory.
Remark: Hence the instrument stand calibrated with respective to
  1. Precision of injection 
  2. Detector performance 
  3. Linearity of responses
  4. Pump performance 

Troubleshooting:

column peak split and double peak problem:

  1. Column washing proper with water.
  2. Check mobile phase pH and proper mixing and degases.
  3. Check proper preparation of test solution.
  4. Check column history for uses.
  5. Last column direction change.

Common problem of Detector:

  1. Noisy baseline.
  2. Drifting baseline.
  3. No response.
  4. Poor sensitivity.
  5. Auto zero not possible.

Common problem of Pump:

  1. Air bubble
  2. Particular mattered
  3.  Pump seal wear

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