Spectrophotometry Spectrophotometer DR CHATCHAWAN SRISAWAT BIOCHEMICAL ANALYSIS Qualitative

��������� (Spectrophotometry ( �������� Spectrophotometer DR. CHATCHAWAN SRISAWAT

BIOCHEMICAL ANALYSIS • Qualitative �������� ( ( to identify the components of a substance or mixture. e. g. Barfoed’s test (test for monosaccharides) xylose glucose fructose lactose )monosaccharide( )disaccharide( Commassie blue test (test for proteins) Negative sucrose Positive

BIOCHEMICAL ANALYSIS • Quantitative �������� ( ( to determine the amounts or proportions of the components of a substance. e. g. ���������� = 105 mg/dl ���������� = 7. 2 g/dl Gravimetry )������� ( Accurate but may not be practical!

��������� (Spectrophotometry ( ������� Io hn I Io = light intensity �������� I = light intensity ������ (absorbance���� optical density [O. D. ]) = log Io/I Absorbance ���� 0 ��������� (I = Io) Absorbance > 0 ���������� (I < Io)

������� spectrophotometer Spectronic 20 - analog reading

������� spectrophotometer 2. Set zero % transmittance Io hn I Transmittance = I Io Absorbance (O. D. ) = log (Io/I)

������� spectrophotometer 4. Insert blank ����� blank ���� cuvette ������� cuvette holder

������� spectrophotometer 4 -5. Insert blank - set 0 absorbance or 100% transmittance (set full scale) blank Io unknown I hn Io I hn ����� biuret + protein

������� spectrophotometer 6 -7. Insert unknown - Read absorbance

������� spectrophotometer Thermo Spectronic - digital reading

������� spectrophotometer Wavelength adjustment knob ������� /���� set zero absorbance (set full scale(

������� spectrophotometer 1. Turn on - warm up 15 min 2. Set wavelength * ������� Thermospectronic ������� set zero transmittance ������� Spectronic 20

������� spectrophotometer 3 - 4. Insert blank and set zero absorbance (set full scale) 5 -6. Insert sample and read absorbance