Lab 1 Flashcards
Spectrophotometry
measurement of light absorbance, identification, quantification, enzyme catalysed rxn measure
X rays
subvalence electrons excited to higher level
visible
valence elecs excited to higher energy levels
IR
molecular vibration
nanometer
10-9 m
Angstrom
10-10 m
c=
Av
wave number
1/A
red light
650nm
entrance slit
focus intense beam of light
exit slit
select wavelength
sample holder
pathlength 1cm
amt of light absorbed depends on
thickness, conc & chemical nature of absorbing compound
A= log lo/I
lo=incident light I=transmitted light
A=ecl
abs= molar conc, conc, pathlength
E
extinction coefficient
abs conc up to
OD3.0
Uses of spectrophotometry
Det prot conc direct/indirect, enzyme activity, DNA quantitation, cell numb of bacteria, chem detection of carbs
protein quantitation methods
UV absorption and colorimetric assay
protein quantitation factors
amt of protein available, presence of interfering cmpds, protein conc, assay specificity, easy and reliability of each compound
protein quantitation
estimate amt of purified protein present after purification
UV absorption methods
reagent or incubation not needed. Measure abs and get std curve
protein conc mg/ml
1.55A280nm-0.76A260nm
200-210nm abs
peptide bond
