Calibration of High Performance Liquid
Chromatography
(Quaternary & Binary)
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)
4.1.2
Water (HPLC Grade)
4.1.3 Acetone
(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
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 :
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
Area of blank
%
Carryover = ------------------------- X 100
Area of sample
Acceptance
Criteria: The % carryover should not exceed 0.02%.
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