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Table - 1
HPLC Operating Conditions and Settings
Operating conditions
Setting
Flow Rate
1.3ml/min
Mobile Phase Temperature
Ambient
Column temperature
35
0
C
Detector Wavelength
195nm
Injection Volume
10 µl
For best precision a fixed volume sample loop is preferred to syringe injection of samples and standards.
To analyze fertilizer solution for urea, allocate 14 minutes for each injection, 12 minutes for run time
and 2 minutes for post run time. For more complex fertilizer solutions, allocate a total of 43 minutes for
each injection, which includes 23 min of run time and 20 min of post-run timeto avoid overlapping.
(b)
HPLC Column
– The chromatography column is a 4.6 mm ID x 250 mm length amino propyl (NH
2
)
column, 5µ particle size. Examples are Phenomenex amino propyl column Spherex, Part # 00G-00051-
E0, or Thermo Scientific Hypersil APS-2 column, Part # 30703-254630. Before use, new columns must be
conditioned as described in Section (c) hereof.
(c)
If the HPLC column is new or has not been in service for about a week or more, it must be
conditioned as follows before using. A conditioned column will usually last for about a year depending
on the number of analyses. This conditioning step is only applied to new columns or columns that are
not used on a weekly basis:
(1)
Using the HPLC instrument, wash the column for about two to four hours at room temperature
with HPLC-grade isopropanol at a flow rate which will maintain at least 200 bars column back
pressure (typically about 1 ml/min).
(2)
Follow step 1 with a second column wash with 100% HPLC-grade acetonitrile at room
temperature for 4 hours.
(3)
Follow step 2 with a final column wash using mobile phase solution [see C(a)] at 1.3 ml/min and
normal analytical operating conditions. This wash should continue for 2 hours or as long
thereafter as needed to obtain a stable base line.
C.
Reagents
(a)
Mobile Phase
– 85% (v/v) acetonitrile with water. Use LC grade acetonitrile having 190 nm
maximum UV cutoff and LC grade water.
(b)
Urea standard-ACS reagent, 99-100.5%, Sigma-Aldrich Chemical Company, Milwaukee, WI.
D
.
External Standard
Urea Standard - Accurately weigh amounts of 100% pure urea of approximately 0.0150g and
0.0300g into separate 100 ml volumetric flasks. Use ultrasound for three minutes to dissolve
and then dilute to volume with mobile phase solution. Shake well.
E. Sample Preparation