Monosaccharides Derivatives to Avoid the

Usage of Refractive Index Detector (RID)

RID
Baseline noise
Not compatible with gradient elution
Low sensitivity
Low efficiencies
Low stability
 Sugar-derivatives
Aldose sugar (glucose, galactose, mannose)
Chromophore Substance
Ketose sugar (fructose) (absorb light at higher wavelength)

Derivatizing Agent :
- PMP (1-phenyl-3-methyl-5-pyrazolone)
- ABEE (p-Aminobenzoic Acid Ethyl Ester)
 A Thesis by Wang (2017)
OPTIMIZATION OF HPLC DETECTION OF PMP DERIVATIVES
OF CARBOHYDRATES
Operating Condition :
- SPD-M10A DAD
- Column : Eclipse Plus C18 Column (4,6 x 250 mm, Agilent)
- Gradient Elution : Acetonitrile 20-30% (45 menit), kembali ke 20%
pada 10 menit kemudian.
- Flow Rate : 0,3 ml/min
- Column Oven Temp : 30 C
 Standar :
- 46,5; 142,5; 285 dan 570 ppm
Reaktan :
- Timbang 87 mg PMP Bubuk, campurkan dengan 1 ml methanol 0,5 M
PMP-Methanol
- 6 gram NaOH + 500 ml air 0,3 M NaOH
- 37% HCl 0,3 M HCl
Mobile Phase :
- Ammonium Acetate (7,7 gr/l) diatur pH-nya hingga 5,51 dengan asam
asetat.
- HPLC Grade Acetonitrile
PMP-derivatives :
- 100 l standar + 100 l 0,3 M NaOH
- Sesuaikan sampai pH 13 dengan 0,3 M NaOH
- Tambahkan 100 l 0,5 M PMP-methanol; Aduk selama 1 menit.
- Panaskan dengan waterbath pada suhu 70 C selama 2 jam.
- Netralkan dengan 100 l 0,3 M HCl
- Tambahkan 4 ml Chloroform
- Aduk 1 menit, diamkan beberapa saat. Lapisan atas (water-
methanol) dipisahkan.
- Saring dengan 0,45 m filter.
(Fang et al., 2015)