Application of UV/Vis
Spetrophotometry Methode:
SMK Negeri 13 Bandung
UV-Vis area
Near UV area
: 200-400 nm
Vacuum UV area : 100-200 nm
Visible area
: 400-700 nm
UV/Vis usage
Purity and identity test in farmacopeia
monography
Content determination (main use):
(1) One-point method: external
standard, standar addition
(2) Multi-point method, calibration
curve: external standard, standar
addition
(3) Single substance or multi
component sample
As detector: HPLC, CE
Purity and identity test
Measurement of UV/Vis spectrum of a
substance refer to reference spectrum
or literature
Maximum wavelength
Determination of absorptivity at
maximum wavelength
Determination of content
(Quantitative analysis)
 Relative method needs reference standard
 Preparation of standard solution (Lambert-Beer‘s law,
about 10 ug/ml)
 For one-point method only needed 1 standard
solution, the concentration should be near the
sample concentration
 For multi-point method (calibration curve ):
differential dilution, calibration curve concentration
vs absorbance
 For multi-point method (standard addition): constant
sample, differential standard addition, linear regretion
of standard concentration added vs absorbance
Solvent
water
Absorption below
(nm)
One Point Method
Cs = ( As / Ab) x Cb
Cs = Sample concentration
As = Sample Absorbance
Ab = Standard Absorbance
Cb = Standard concentration
Multi Point Method: External Calibration
Sample Absorbansi
c
Multi Point Method: Standard Addition
Analit concentration in
sample solution
UV Spectrum
Lambert-Beer‘s Law






A = Absorbance
e(l) = molar absorptivity
C = concentration [ mole / l]
c = concentration[g/ 100 ml]
b = Cell length (cuvette length [cm])
A1%, 1 cm = Absorptivity jenis
 e(l) is absorbance (A) of a solution (C= 1 mol/ l) whwn cell
length is 1 cm (b= 1) and the wave length is l.
Lambert-Beer‘s validity area
•0,2 - 0,8 absorbance unit (A)
•1 mg/ 100 ml equivalent to 0,2 - 0,8 absorbance unit (A)
•A > 0,8 : interference
•A < 0,2 : worse precision and accuracy
• Smallest fotometric error when A = 0,434
Experiment and calculation
 Prepare 2 standard solution of substance 1 and 2
 Measure the absorbance of substance 1at l1 and l2, count
the a1(l1) and a1(l2)
 Measure the absorbance of substance at l1 and l2, tcount
the a2(l1) and a2(l2)
 Measure the absorbance of sample solution (mixture of
substance1 and 2) at l1 (Al1) and l2 (Al2)
 This equation is valid :
Al1 = a1(l1) c1 + a2(l1) c2
Al2 = a1(l2) c1 + a2(l2) c2
 c1 and c2 could be determined
Influence of pH to spectrum
Spectrum of Methyl and Propilparaben
Methyl Paraben
Propil Paraben
CALIBRATION CURVE OF
METHYL PARABEN
Concentration of methyl
paraben (µg/mL)
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
Absorbance (A)
at 256 nm
0,2841
0,3061
0,3196
0,3785
0,4216
0,4607
0,4987
0,5504
Calibration curve of Methyl paraben
at 256 nm
0.6
Serapan (A)
Regretion equation:
 y = 0,1960 x + 0,1476;
koefisien korelasi (r) = 0,99;
batas deteksi (BD) =
0,3306 mg/mL; batas
kuantisasi (BK) = 1,1020
mg/mL dan koefisien
variasi fungsi regresi (V
xo) = 0,85%
0.5
0.4
0.3
0.2
0.1
0
0
0.5
1
1.5
2
Konsentrasi metil paraben (mg/mL)
2.5
Kalibrasi Spektrofotometer
 Perlu dilakukan untuk mencegah kesalahan
pembacaan panjang gelombang dan absorban
 Kalibrasi skala panjang gelombang: larutan
holmium dioksid dalam asam perklorat.
Perbedaan penunjukkan skala panjang
gelombang pada alat dengan panjang gelombang
seharusnya digunakan untuk mengoreksi
pembacaan alat.
 Kalibrasi skala fotometrik: larutan kalium
bikromat dalam asam sulfat. Berhubungan
dengan intensitas sumber radiasi (life time
sumber radiasi)
Kalibrasi Skala Fotometrik
Kontrolle: Messung von A bzw A(1%, 1 cm). im Bereich von 200
bis 400 nm mit schwefelsaurer K2Cr2O7-Lösung. Für den
Bereich von 400 bis 800 nm gibt das DAB keine Kontrollsubstanz
an. Geeignet wären z.B. Nickelsulfat, Cobaltsulfat,
Kaliumpermanganat.