Spectrophotometry

Week ThreeSpectrophotometry A = bc

Technology and Scale100mm10mm1mm100m10m1m100nm10nm1nm0.1nmUnaided EyeLight MicroscopyElectron MicroscopyAtomic Force Microscopy(0.2 mm)(0.2 m)(0.2 nm)Human HairMost CellsBacteriaVirusesMacromolecules Proteins and DNAAtomsTEM

Technology and ScaleGentile, F. et al. (2012) Nano Lett., 12(12), p6453

SpectrophotometryIdentification of macromoleculesDisease Diagnosis (ELISA)Mass spectrophotometry (MS)

Concentrations of substancesBreathalyzer (BAC)DNA and Protein (UV)

Structure and functionNMR, Ramen, and UV circular dichroismImmunofluorescence Assay

He 36 hpi Eg1014.5x 488 (1.20s DO38g[25])4.2x 647 (0.25s DO38g[00]) 7.9x 555 (0.25s DO65g[15])Scale Bar 11.00 m

Later stages of NS3 staining are perfectly perinuclear and associated with dsRNA. ABCF3 is usually not associated, as seen here. The ABCF3 staining appears to be on the periphery of the NS3 staining.

PrinciplePhoton with energy equal to difference between electron energy levels

PrinciplePhoton with energy equal to difference between electron energy levelsElectron jumps levels (n=2 n=3)

PrinciplePhoton with energy equal to difference between electron energy levelsElectron jumps levels (n=2 n=3)Excited electron loses energy as a photon and moves back down (n=3 n=2)

Principle[X] = 0.1 M[X] = 5 M

PrincipleConcentration [X]Absorbance[X] = 0.1 M[X] = 5 MAbsorbanceConcentration [X]AbsorbanceConcentration [X]AbsorbanceAbsorbanceAbsorbanceAbsorbanceConcentration [X]Absorbance

Principle extinction coefficientConcentration [X]Absorbance

A= bc Lambert Beer Law (Beers Law) A = -log10 I1/I0

OD

= A/bc bc

Spectrophotometer

Application

ApplicationDell, E.J. et al.(2008) BMG Labtech, App Note169DNA absorbs optimally at 260 nmTherefore.?

ApplicationProteins absorb optimally around 280 nmProblem?

ApplicationDNA Purity 260/2801.7 Good1.8-1.9Pure!1.95 Something aint right!DNA 260=0.020 (g/mL)-1cm-1OD260* 50 g/mL

ApplicationBradford Assay= 595 nm FCF = 625 nm

Todays Lab WorkCiVi=CfVfCi=UnknownCf=Diluted

Vf/Vi=Dilution Factor

Chart2

0.1

0.25

0.3

0.35

0.4

0.5

0.7

0.8

0.9

1

1.1

1.2

Concentration [M]

Absorbance (OD)

FCF Standard Curve

Sheet1

0.10.065

0.250.1625

0.30.195

0.350.2275

0.40.26

0.50.325

0.70.455

0.80.52

0.90.585

10.65

1.10.715

1.20.845

Sheet1

Concentration [M]

Absorbance (OD)

FCF Standard Curve

Sheet2

Sheet3

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