# Calibration of High Performance Liquid Chromatography

Contents

## Calibration of High Performance Liquid Chromatography

1.0 PURPOSE

The purpose of the standard operating procedure is to calibrate the High Performance Liquid  Chromatography (Quaternary & Binary HPLC).

2.0  SCOPE

Standard operating procedure is applicable to the staff responsible for the operation and calibration of the High Performance Liquid Chromatography (Quaternary & Binary).

3.0   RESPONSIBILITY

3.1    Section Incharge

3.2    Technical Manager

3.3    Q.A. Manager

4.0   PROCEDURE

4.1   MATERIAL REQUIRED

4.1.1   Instrument under calibration (Quaternary & Binary HPLC)

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

4.2        CALIBRATION

4.2.1.    CALIBRATION OF PUMP

4.2.1.1  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).

4.2.1.2 Connect the pump output to a restrictor coil. Set the pump flow rate to 1 ml/min. Pressure will be 900-1200 psi of backpressure.

4.2.1.3 Start the pump and let the pressure stabilize.

4.2.1.4 Observe the minimum and maximum pressure during the 1 min interval.

4.2.1.5 Determine the minimum and maximum pressures observed as well as the delta, or difference between observed values. Determine the mid point pressure (MP) between the maximum and the minimum, 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.

4.2.1.1 FLOW ACCURACY TEST

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

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

4.2.2.1 Connect the backpressure regulator to the output of the pump. The backpressure as read on the pump will be between 900-1200 psi.

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

4.2.2.3 Start the pump and fill the lines with water.

4.2.2.4 Set the pump flow at 1 ml/min.

4.2.2.5 When pressure stabilizes, direct the flow of water into the beaker and simultaneously start the stopwatch.

4.2.2.6 Let the pump run for 5 min.

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

4.2.2.8 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.

4.2.3 CHECKING FLOW AT 3 ML/MIN (HIGH FLOW RATE)

4.2.3.1 Connect the backpressure regulator to the output of the pump. The backpressure as read on the pump will be between 900-1200 psi.

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

4.2.3.3 Start the pump and fill the lines with water.

4.2.3.4 Set the pump flow at 3 ml/min.

4.2.3.5 When pressure stabilizes, direct the flow of water into the beaker and simultaneously start the stopwatch.

4.2.3.6 Let the pump run for 5 min.

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

4..2.3.8 Subtract the final weight with the initial weight of the beaker (W2-W1) & divide the weight of the water by 15 & with the water density to get the volume delivered per minute.

Acceptance Criteria: The volume of water should be ±0.5 ml

4.3 COMPOSITION ACCURACY TEST

The Composition Accuracy Test proves that the proportioning valves are operating correctly. This is accomplished by varying the amounts of solvent entering the pump through the proportioning valves. Using acetone solution, which has absorbance at 265 nm as one solvent, and water, which does not show any absorbance at 265 nm.

4.3.1 Fill reservoirs A and C with water.

4.3.2 In a 500 ml graduated cylinder, accurately pipette 1.0 ml of acetone into 250 ml of HPLC- grade water. Dilute to 500 ml with HPLC grade water. This is the 0.2% Acetone Solution.

4.3.3 Place acetone solution into reservoir B and D.

4.3.4 Degas all mobile phases for 5 min.

4.3.5 Purge all reservoirs with 30 ml of solvent.

4.3.6 Bypass the column.

4.3.7 Set the detector wavelength to 265 nm.

4.3.8 Pump the acetone solution for 30 min through channel B and 30 min through channel D if applicable. Check the absorbance level is 0.5-0.7 AU.

4.3.9 Ensure the pump display reads at least 1000± 200 psi of backpressure at 5 ml/min.

4.3.10 Generate and run the method in table 1 and 2, zeroing the detector at the end of step zero.

Table 1: For Binary and quaternary (Reservoirs A and B)

STEP             TIME            FLOW           % A              % B              CURV           AU

0                     5.0                5.0                100               0                   0.0

1                     1.0                5.0                100               0                   0.0

2                     3.0                5.0                0                   100               0.0               AU1=

3                     3.0                5.0                10                 90                 0.0               AU2=

4                     3.0                5.0                49                 51                 0.0               AU3=

5                     3.0                5.0                51                 49                 0.0               AU4=

6                     3.0                5.0                90                 10                 0.0               AU5=

Table 2: For Quaternary only (Reservoirs C and D)

STEP             TIME            FLOW           % A              % B              CURV           AU

0                     5.0                5.0                100               0                   0.0

1                     1.0                5.0                100               0                   0.0

2                     3.0                5.0                0                   100               0.0                AU6=

3                     3.0                5.0                10                 90                 0.0                AU7=

4                     3.0                5.0                49                 51                 0.0                AU8=

5                     3.0                5.0                51                 49                 0.0                AU9=

6                     3.0                5.0                90                 10                 0.0                AU10=

Compute the actual compositions (C).

% B in A (Binary and Quaternary)                           % D in C (Quaternary only)

C (90) = AU2/AU1 ´ 100%                                      C (90) = AU2/AU6 ´ 100%

C (51) = AU2/AU1 ´ 100%                                      C (51) = AU2/AU6 ´ 100%

C (49) = AU2/AU1 ´ 100%                                      C (49) = AU2/AU6 ´ 100%

C (10) = AU2/AU1 ´ 100%                                      C (10) = AU2/AU6 ´ 100%

Acceptance Criteria: The actual composition value should be within 1.0 % of the target %.

4.4. CALIBRATION OF DETECTOR

4.4.1 CAFFEINE WAVELENGTH VERIFICATION

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

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

4.4.1.2 In another reservoir place a bottle of HPLC water. Using the HPLC water, purge the pump lines using prime purge valve. Connect outlet of pump directly to inlet of the UV/VIS flow cell.

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

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

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

4.4.1.6 Set the wavelength to 205 nm.

4.4.1.7 Start pump at 1 ml/min with dilute caffeine solution. After 5 minutes check that absorbance reading is between 0.9 and 1.3 AU.

4.4.1.8 Record the observed wavelength maximum. Record this value in the instrument performance

CHANNEL PARAMETERS

Run Time                  :   23.00min

Sampling Rate          :    2.0000 pts/s

DETECTOR PARAMETERS

Step    Time   Wavelength     Auto zero   Step    Time  Wavelength Auto zero

1           1.0             300                  No          11          1.0            269                 No

2           4.0             200                  No          12          1.0            270                 No

3           1.0             201                  No          13          1.0            271                 No

4           1.0             202                  No          14          1.0            272                 No

5           1.0             203                  No          15          1.0            273                 No

6           1.0             204                  No          16          1.0            274                 No

7           1.0             205                  No          17          1.0            275                 No

8           1.0             206                  No          18          1.0            276                 No

9           1.0             207                  No          19          1.0            277                 No

10         1.0             208                  No          20          1.0            278                 No

PUMP PARAMETERS

Step           Time         Flow           A              B             C             D           Curve

0                1.0            3.00        100.0       0.0           0.0         100.0          0.0

1                23.0          3.00          0.0         0.0           0.0         100.0          0.0

Acceptance Criteria: The wavelength maximum at 205 nm and/or 273 nm should be in the range of ±1 nm.

4.5. CALIBRATION OF COLUMN OVEN

4.5.1 TEMPERATURE ACCURACY

Measure oven temperature accuracy at three points in the normal operating range of 5°C above ambient to 100°C.

4.5.1.1 Remove the oven door cover. 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.

4.5.1.2 Ensure the oven is installed properly and power switched on at the Column Oven. Select a Set temperature of 20°C for the pelteir oven or 30°C via the oven’s keypad for the heat only oven.

4.5.1.3 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.

4.5.1.4 Repeat step (c) for a Set Temperature of 40°C for pelteir oven or 50°C for the Heat only oven.

4.5.1.5 Perform the Temperature Stability Test.

4.5.1.6 Repeat step(c) for a Set temperature of 60°C for the pelteir oven or 70°C for the Heat only oven.

Acceptance Criteria: The value should be within ±1°C.

4.5.2 TEMPERATURE STABILITY

Measure oven stability at 40°C.

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.

4.6  CALIBRATION OF AUTOSAMPLER:

The initial system set up conditions:

Wavelength                 : 260 nm

Flow Rate                    : 1.0 ml/min

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

Flush Solvent              : 80% Methanol/Water

Column                       : C18

Dilution Medium          : Mobile Phase

4.6.1 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 Autosampler can repeatedly inject the same volume of sample numerous times.

Inject 6 injections of 10 mL each. This test also checks that the Autosampler can accurately inject sample volumes from multiple positions on the tray.

ACCEPTANCE CRITERIA:

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

4.6.1.1 LINEARITY:

Linearity will check that the Autosampler can accurately inject varied amounts of sample and that the injected volumes are correct. We do this by injecting multiple samples of different injection volumes.

The injection volumes are 4 injections of 5 mL from vial at position 1, 6 injections of 10 mL from vial 10, and finally 4 injections of 20 mL from vial 100. There should be linear relationship between sample volume and area under the peak.

Acceptance Criteria: The R-squared value should be of 0.999 or greater

4.6.1.2 CARRYOVER

Carryover is the amount of previous sample retained by the system and therefore has a presence in the current sample. Looking at the peak of Naphthalene and at the mobile phase blank checks carryover. There should be very little if any of the Naphthalene in the mobile phase blank at the retention time of the Naphthalene peak. The specification for this test is less than or equal to 0.02%.

Area of blank

% Carryover = ————————- X 100

Area of sample

Acceptance Criteria: The % carryover should not exceed 0.02%.

## FAQs

1. How often should HPLC calibration be performed?
• Calibration frequency depends on usage and industry requirements. However, it’s generally recommended to calibrate HPLC systems at regular intervals, often monthly.
2. Are automated calibration systems worth the investment?
• Yes, automated calibration systems offer increased efficiency and accuracy, making them a worthwhile investment for laboratories seeking optimal performance.
3. Can calibration be customized for specific industry needs?
• Absolutely. Calibration guidelines can be tailored to meet the unique requirements of different industries, ensuring compliance and accuracy.
4. What are the consequences of neglecting HPLC calibration?
• Neglecting calibration can lead to inaccurate results, compromising the integrity of analytical data. It may also result in non-compliance with industry regulations.
5. How can calibration contribute to environmental sustainability?
• Implementing sustainable calibration practices, such as reducing waste and energy consumption, contributes to environmental sustainability by minimizing the impact of HPLC calibration on the environment.

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