Calibration of High Performance Liquid Chromatography (isocratic)

 Calibration of High Performance Liquid
Chromatography (isocratic)

1.0
      PURPOSE

The purpose of the standard operating procedure is
to calibrate the High Performance

Liquid Chromatography (Isocratic HPLC).

2.0
      SCOPE     

Standard
operating operation is applicable to the staff responsible for the operation
and

calibration
of the High Performance Liquid
Chromatography (Isocratic)
.

 

3.0       RESPONSIBILITY

3.1    Section Incharge

3.2    Technical Manager

3.3    Q.A. Manager

 

 4.0       PROCEDURE

 

4.1       MATERIALS
REQUIRED

4.1.1    Instrument
under calibration.(Isocratic HPLC)

4.1.2    Acetone
(HPLC Grade)

4.1.3
   Water
(HPLC Grade)

4.1.4
   Caffeine
(Working Standard)

4.1.5
   Calibrated
Digital Thermometer

4.1.6
   Naphthalene

4.1.7
   Calibrated
Glassware

4.1.8
   Weighing
Balance

5.0         PROCEDURE

5.1         CALIBRAION OF PUMP

5.1.1      PRESSURE
PULSATIONS TEST

  The Pressure Pulsations Test is to verify
that the output flow fluctuations are within      

  specification (2% peak to peak of the running
pressure).

 

5.1.1.1   Set the pump flow rate to 1 ml/min.

5.1.1.2   Start the pump and let the pressure
stabilize.

5.1.1.3   Observe the minimum and maximum pressure
during the 1 min interval

 

 

 5.1.1.4 Determine the minimum and maximum
pressures observed, or difference between them. Determine   the mid point pressure (MP) between the max
and min, this is the mid point pressure used to compute 2 % peak-to-peak error
band.

ACCEPTANCE CRITERIA:

 Difference between the minimum and the maximum
pressure should be less than 2% of the   

 mid point pressure.

 

5.1.2      FLOW
ACCURACY TEST

This
test is used to prove that the flow rate selected on the pump is delivered in
the correct time.

 

CHECKING FLOW AT 1 ML/MIN
(LOW FLOW RATE)

5.1.2.1
 Connect the backpressure regulator to
the output of the pump.

5.1.2.2  Zero the
scale of the weighing balance. Place a 50 ml beaker on a scale and note the    

              reading
of the empty beaker (W1).

5.1.2.3  Start the pump and fill the lines with water.

5.1.2.4  Set the pump flow at 1 ml/min.

5.1.2.5  When
pressure stabilizes, direct the flow of water into the beaker and
simultaneously   

             start
the stopwatch.

5.1.2.6
 Let the pump run for 5 min.

5.1.2.7
 Remove the beaker after 5 min and weigh
the beaker containing water (W2).

5.1.2.8  Subtract
the final weight with the initial weight of the beaker (W2-W1) & divide the
 

Weight
of the water by 5 & with the water density to get the volume delivered per   minute.

5.1.2.9
 Repeat the above procedure for 10 min.             

 

     5.1.3      CHECKING FLOW AT 2 ML/MIN (HIGH FLOW RATE)

 

5.1.3.1
  Connect
the backpressure regulator to the output of the pump.

5.1.3.2   Zero
the scale of the weighing balance. Place a 50 ml beaker on a scale and note the
reading of the   empty beaker (W1).

5.1.3.3   Start
the pump and fill the lines with water.

5.1.3.4
  Set
the pump flow at 2 ml/min.

5.1.3.5   When pressure stabilizes, direct the flow of
water into the beaker and simultaneously  

               start
the  stopwatch.

5.1.3.6
   Let
the pump run for 5 min.

5.1.3.7    Remove
the beaker after 5 min and weigh the beaker containing water (W2).

5.1.3.8 
 Subtract the final weight with
the initial weight of the beaker (W2-W1) & divide the   

             
weight of the water by 10 & with the water density to get the volume
delivered per minute.

5.1.3.9    Repeat
the above procedure for 10 min.

ACCEPTANCE CRITERIA:

The
volume of water collected should be ±0.5 ml.

 

5.2.    CALIBRATION
OF DETECTOR

 

5.2.1      CAFFEINE
WAVELENGTH VERIFICATION AND ACCURACY

Dissolve
10 mg of caffeine WS with 1000 ml of water. Dissolve and mix completely.

 

5.2.1.1   Place
the 1-liter of dilute caffeine mixture on an available reservoir of the pump.

5.2.1.2   In another reservoir place a bottle of HPLC
water. Using the HPLC water, purge the    

              pump
lines. Connect outlet of pump directly to inlet of the UV/VIS flow cell.

5.2.1.3   Set
the UV/VIS Detector wavelength to 300 nm.

5.2.1.4   Start
the pump flow rate at 1ml/min with water and pump for 5 minutes.

5.2.1.5   Auto
zero the detector with water in the flow cell.

5.2.1.6   Set
the wavelength to 205 nm.

5.2.1.7
  Start pump at 1 ml/min with dilute
caffeine solution. After 5 minutes check the

  absorbance.

5.2.1.8
  Record the observed wavelength maximum.

 

5.2.2     CHANNEL PARAMETERS

              Run Time                  :   23.00min

              Sampling Rate          :   
2.0000 pts/s

 

5.2.3
DETECTOR PARAMETERS

 Step    Time   Wavelength     Auto zero  

1           1.0             300                  No          

2           4.0             200                  No         

3           1.0             201                  No         

4           1.0             202                  No         

5           1.0             203                  No         

6
          1.0             204                  No         

7           1.0             205                  No         

8           1.0             206                  No         

9           1.0             207                  No         

10         1.0            
208                  No         

11         1.0             269                  No

12         1.0             270                  No

13         1.0             271                  No

14         1.0             272                  No

15         1.0             273                  No

16         1.0             274                  No

17         1.0             275                  No

18        
1.0             276                  No

19        
1.0             277                  No

20        
1.0             278                  No

 

ACCEPTANCE CRITERIA:
The wavelength maximum at 205 nm and 273 nm should be in the range of
±1 nm.

 

5.3. CALIBRATION OF COLUMN OVEN

 

5.3.1      TEMPERATURE
ACCURACY

 

 Measure oven temperature accuracy at three
points in the normal operating range of  

  5°C above ambient to 100°C.

 

5.3.1.1 
Open the oven door. Carefully place the probe of the thermometer in the
column holder   

              bracket
so the tip of the probe is in the air stream of the oven.

5.3.1.2  Ensure the oven is installed properly and
power switched on at the Column Oven.  

             Select
a Set Temperature of 40
°C
for the heat only oven.

5.3.1.3  Monitor
the reported oven temperature (on the oven’s display) vs. the temperature  

             record
on the thermometer. Allow the system to reach the set point and to stabilize
for  

             15- 30
minutes. Record the temperature displayed on the digital thermometer.

5.3.1.4  Repeat step (c) for a Set Temperature of 60°C, 80°C & 100°C for the oven.

                                                                                        

ACCEPTANCE CRITERIA:
The value should be within ±1°C.

 

5.3.2      TEMPERATURE
STABILITY

 

5.3.2.1 
With the set temperature set to 40
°C
and stabilized, take six further readings of the Digital Thermometer at
fifteen-minute intervals. Record the temperature range. The range of the
results should be less 1
°C.

 

ACCEPTANCE CRITERIA:
The value should be Max (reading) – Min (reading) <1°C.

 

5.4. CALIBRATION OF INJECTOR:

The
initial system set up conditions:

Wavelength              : 260 nm

Flow
Rate                 : 1.0 ml/min

Mobile
Phase                       : Methanol:
Water (80:20 v/v)

Column                    : C18

Dilution
Medium       : Mobile
Phase

 

5.4.1      LINEARITY:

Linearity
will check that the injector can accurately inject varied concentration of
sample. We do this by injecting different concentration of Naphthalene of same
injection volumes.

The
different concentrations are 10 ppm, 20 ppm, 30 ppm 40 ppm & 50 ppm. There
should be linear relationship between the concentration of the sample and area
under the peak.

            ACCEPTANCE CRITERIA:
The R-squared value should be of 0.999 or greater.

 

5.4.2    REPEATABILITY (PRECISION)

Repeatability
will look at the standard deviation of the area of the Naphthalene peak, the
next to last peak in the chromatogram to check Repeatability. The Naphthalene
peak from each data file will be averaged and the standard deviation
determined. From the standard deviation the RSD is calculated. The purpose of
Repeatability is to verify that the injector can repeatedly inject the same
volume of sample numerous times.

Inject
6 injections of 20
mL
each.

 

        
 ACCEPTANCE CRITERIA
:
The standard deviation should be less than or equal to 0.5 %.

 

 

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