HPLC

Glyphosate
CE
CE-UV ( Kodama et al., 54(5) 602- 606, 2008) Journal of Health Science
1) Instrument
Bare fused-silica capillary column with 50 m internal diameter and variable lengths (58-75 cm)
Orthogonal flow and isocratic
2) Samples and standard solutions
Prepare stock glyphosate (100 ppm) 10 mg in 100 mL acetonitrile-water (10:90 v/v) ~
weight 0.05 g in solvent (20 mL acetonitrile and 180 mL pure water)
Since the maximum allowable concentration of Glyphosate in drinking water is 280 g/L,
the standard conc range is (50,100,200,400 & 800) ppb
100 ppm
10 ppm
m1v1= m2v2
(100 ppm)(v1) = (10 ppm)(50 mL)
V1= 5 mL
10 ppm
1 ppm
m1v1 = m2v2
(10 ppm) (v1) = (1 ppm)(100 mL)
V1= 10 mL
1000 ppb
800 ppb
m1v1 = m2v2
(1000 ppb) (v1) = (800 ppb)(5 mL)
V1= 4 mL , + 1 mL solvent
1000 ppb
400 ppb
m1v1 = m2v2
(1000 ppb) (v1) = (400 ppb)(5 mL)
V1= 2 mL , + 3 mL solvent
1000 ppb
200 ppb
m1v1 = m2v2
(1000 ppb) (v1) = (200 ppb)(5 mL)
V1= 1 mL , + 4 mL solvent
1000 ppb
100 ppb
m1v1 = m2v2
(1000 ppb) (v1) = (100 ppb)(5 mL)
V1= 500 L , + 4500 L solvent

1000 ppb
50 ppb
m1v1 = m2v2
(1000 ppb) (v1) = (50 ppb)(5 mL)
V1= 250 L , + 4750 L solvent
3) Experimental Conditions Preparation

Glyphosate separation --- BGE--- 40 mM acetate buffer (pH 5.0) containing 5mM
CuSO4
Acetate Buffer Preparation
100 mM , pH 5.0
Acetic acid : 3.55 mL , Sodium Acetate : 529.3 mg in 1 L deionized water.
100 mM
40 mM
C1v1 = C2v2
(100 mM) (v1) = (40 mM)(50 mL)
V1= 20 mL
5 mM CuSO4
= 5 m = 0.005 mol/L x 159.609 g/mol
= 0.798 g/L = 0.0798 g CuSO4 in 100 mL water

Conditioned the capillary

1 M HCl for 10 mins
Water for 3 mins
BGE for 5 mins
Sample solution and the BGE were injected at pressure of 50 mbar for 3
sec, respectively
Between runs, the capillary was flushed with BGE for 5 mins. (the
capillary was kept at 25C)
The power supply was operated in the constant-voltage mode at +15kV.
At the end of each day, the capillary was flushed with water for 5 min,
methanol for 5 min and air for 5 min.

Result

2) CE-UV (Chang, 2005 Journal of Chinese Chemical Society, 52,785-792)

CE system
Capillary used for separation 50m ID x 360m OD x 57 cm total length , effective
length 45 cm.
Before run, the capillary was washed with CE-Buffer for 5 mins.
The capillary was equilibrated with the buffer under an electric field of 368 V/cm for 1
hour.
Sample were injected by raising the anodic end 18 cm above its normal position for 10480 s.
UV absorbance detection was performed at 260 nm with a commercial UV absorbance
detector.

Derivatization Procedure
50 L of standard were mixed with 10L of 25 mM borate buffer (pH 9.5).
140 L FMOC-Cl (4 mM in acetonitrile) was then added and mixed thoroughly.
The resulting solution were shaken for 30 min using a vortex shaker.
Preparation of 25 mM borate buffer (buat 2 set, satu tambah 200 mM NaCl)
= 0.025 mol/L x 381.37 g/mol
= 9.53 g/L = 0.953 g of borax in 100 mL VF, added water , heated at 60C and stir. Check
and adjust the pH if needed. (pH 9.5)
200 mM NaCl
= 0.2 mol/L x 58.44 g/mol
= 11.68 g/L = 1.168 g in 100 mL VF of water
Preparation of 4 mM FMOC-Cl in acetonitrile
= 0.004 mol/L x 258.699 g/mol
= 1.03 g/L = 0.103 g of FMOC-Cl in 100 mL VF, added acetonitrile