Purpose:
The purpose of this SOP is to provide written
guidelines for the Calibration of High
performance Liquid Chromatography
Calibration of Pump :
Flow Test :
Check the flow at the out let of the detector with
column in a standard
measuring
cylinder to verify the flow.
Connect C18 column with 0.45 filtered Distilled water
as mobile phase.
Check the flow for 0.5 ml/min, 1.5ml/min & 2ml/min.
Limit : ± 1%
Frequency: Once in a month.
Pressure Linearity :
Check the pressure at 0.5 ml/min in Kgf and then check the corresponding pressure at
1.0, 1.5, 2.0 and 2.5 ml/min and observe the pressure in Kgf.
Limit : ± 10 % of actual pressure.
Frequency: Once in a month.
System Suitability :
(Ref : IP 1996)
Check for relative standard deviation on daily basis/as
& when required on
three replicate injections. (as per BP) (Limit NMT 2%)
Determination of Column Efficiency :
Purge the HPLC system with solvent like distilled
water to remove the traces of impurities
present in the tubing & Connecting parts, if any. Cleaning of injection port & syringe is
also necessary. Make sure that the whole system is perfectly cleaned.
Connect the column with the direction of flow arrow
pointing towards detector, if mentioned on column. For a new column, first give
slow washings by solvents like Acetonitrile, Methanol (HPLC Grade) &
distilled water filtered through 0.2
membrane filter. Flow rate is increased gradually from 0.5ml/min. up to
1.5 ml/min. increments. Thus equilibrate the column when it is 1st installed.
 |
| Determination of Column Efficiency |
Where
X =
Arithmetic mean of set
Xi = Individual measurement in a set of N
measurement
Determination of Column
Efficiency :
Perform an efficiency test on the new column by
following procedure :
Prepare mobile phase with the combination of 70:30
(Methanol & water). Prepare mobile phase freshly on the day of use. Degas
the mobile phase.
Chromatographic conditions :
= 254 nm
Test Solution - In a 50 ml volumetric flask, take 1.0
ml of Benzene then volume make up to mark with the methanol. Pipette out 5.0 ml
in 50 ml volumetric flask and volume make upto the mark with methanol.
Equilibrate the column with mobile phase till the
detector displays constant absorbance or Zero absorbance. Column saturation can
be checked 00. 0 Pump absorbance should be zero.
1st check the absence of impurities in mobile phase by
injection it through the column. A straight line printed on chromatogram shows
that there is no any impurity present in mobile phase
or in the column. i.e. stationary phase.
Now inject the filtered test solution after
satisfactory separation of the components from mix. Chromatogram draws the
straight line passing through the peak center. Measure the width of the peak at
it's half height. Generally this calculations are done on middle peak, i.e.
benzene peak
Calculate the no. of theoretical plates (N) by using
formula:
Where,
N = 16
x [T/W}2
16
= constant
T
= Retention time of the peak of interest.
W =
Width of peak at it's 5.0 %
height.
Generally theoretical are plates calculated as
plates/meter.
Wash the column with water, acetonitrile & methanol
so as to clean the column free from any traces of test solution. Flushing with
a heat organic solvent is usually sufficient to remove any contaminant. Store
the column in a suitable solvent, which is prescribed by manufacture, or in a
solvent like 60:40 (methanol & water)
Theoretical plates can be calculated periodically or
when required. After the use of column, make sure that the end fittings are
firmly in place which prevents column drying.
Frequency : Initially when a new column is
purchased or Yearly required.
Calibration of the
Detector:
Check the reference energy of the dectetor lamp. The energy of the D2 lamp should not be
less than 730 at 230 nm.
Linearity of the Detector Response, and Auto injector.
and
Uniformity of the Auto Injector is done as follows.
Chromatographic condition as follows
Column
: ODS C18 25cm X 4.6 mm
Mobile Phase :
Methanol : Water(70:30)v/v
Flow Rate
: 1.0 ml /min.
Wavelength :
254.0 nm.
Injection Qty.
: 20 ul.
Prepare 3 different ratios of benzene and tolune in
methanol as given below and inject each solution in triplicate.
Standard Preparation.:
LEVEL 01
Pipette out 0.5 ml of benzene and 1.0 ml of tolune into
50 ml V.F. and dilute with methanol upto mark . Further dilute 5.0 ml to 50 ml
with methanol.
LEVEL 02
Pipette out 1.0 ml of benzene and 1.0 ml of tolune into
50 ml V.F. and dilute with methanol upto mark . Further dilute 5.0 ml to 50 ml
with methanol.
LEVEL 03
Pipette out 1.5 ml of benzene and 1.0 ml of tolune into
50 ml V.F. and dilute with methanol upto mark . Further dilute 5.0 ml to 50 ml
with methanol.
The retention time of benzene & tolune should be
reproducible in each three levels
{Limit ±0.5 mins.}
Calculate the area ratio of benzene to tolune . And
Calculate RSD of each level is NMT 1.0 % . And plot the graph of a) Level verses Area Ratio. B) Peak Area of
Benzene Verses Concentration % v/v C)Peak Area of
Benzene verses no. of injection. D) Peak
Area of Tolune verses No. of injection.
Frequency : Yearly required.
Precautions:
Wash the column thoroughly before and after use with
prescribed solution .
Wash the injection port with water and with prescribed
mobile phase before
use.
Inject the sample only after the system is stabilized
to get accurate results.
Close the instrument as per the sequence described in
above procedure.
Maintain the cleanliness of instrument before &
after use at each time.
