《紫外-可见光谱》课程PPT教学课件（讲稿，英文版）Homoannular Heteroannular

Homoannular Heteroannular CiSoid (transoid 1=214nm Parent 1=253nm F217(acyclic) Increments for Double bond extending conjugation 30 30 Alkyl substituent or ring residue Exocyclic double bond Polar groupings OC(O)CH3 OR Cl.-Br nR2 SR 30 30

Homoannular (cisoid) Heteroannular (transoid) Parent l=253 nm l=214 nm =217 (acyclic) Increments for: Double bond extending conjugation 30 30 Alkyl substituent or ring residue 5 5 Exocyclic double bond 5 5 Polar groupings: -OC(O)CH3 0 0 -OR 6 6 -Cl, -Br 5 5 -NR2 60 60 -SR 30 30

Example 1: Transoid 217nm Alkyl groups or ring residues: 3x 5 15 nm Calculated 232nm Observed 234nm

Example 1: Transoid: 217 nm Alkyl groups or ring residues: 3 x 5 = 15 nm Calculated: 232 nm Observed: 234 nm

Example 2 soid 253nm Alkyl groups or ring residues: 2X 5= 10 nm Calculated 263nm Observed 256nm

Cisoid: 253 nm Alkyl groups or ring residues: 2 x 5 = 10 nm Calculated: 263 nm Observed: 256 nm Example 2:

Example3: Transoid 214nm Alkyl groups ring residues: 3 5- 15 nm Exocyclic double bond n Calculated 234nm Observed 235nm

Transoid: 214 nm Alkyl groups ring residues: 3 x 5 = 15 nm Exocyclic double bond: 5 nm Calculated: 234 nm Observed: 235 nm Example3:

Example 4: Cisoid 253nm Alkyl groups/ ring residues: 4x 5 20 nm Exocyclic double bond 5nm Calculated 278nm Observed 275nm

Cisoid: 253 nm Alkyl groups / ring residues: 4 x 5 = 20 nm Exocyclic double bond: 5 nm Calculated: 278 nm Observed: 275 nm Example 4:

Base values R alkyl or ring residue R入=246nm REH 入=250nn R=OH, OR 入=230nm Increment for each substituent Alkyl or ring residue 3 10 -OH-OR o,m 25 O o m 20 o m 0 10 -Br o,m NH 2 o,m 13 58 NHC(O)CH3 o m 20 NHCH3 N(CH3)2 o,m 85

Base values : R = alkyl or ring residue l = 246 nm R = H l = 250 nm R = OH, OR l = 230 nm Increment for each substituent : -Alkyl or ring residue o,m 3 p 10 -OH, -OR o,m 7 p 25 - O - o,m 11 p 20 -Cl o,m 0 p 10 -Br o,m 2 p 15 -NH 2 o,m 13 p 58 -NHC(O)CH 3 o,m 20 p 45 -NHCH 3 p 73 -N(CH 3 ) 2 o,m 20 p 85

HO HO OH Parent chromophore 230nm p-OH: 25 nm m-OH 27=14nm Calculated 269nm observed 270nm

Parent chromophore: 230 nm p-OH: 25 nm m-OH: 2 x 7 = 14 nm Calculated: 269 nm Observed: 270 nm

Parent chromophore 246nm o-Ring residue: 3 nm m-Bra 2nm Calculated 251nm observed 253nm

Parent chromophore: 246 nm o-Ring residue: 3 nm m-Br: 2 nm Calculated: 251 nm Observed: 253 nm

Protein absorbances come from 3 sources →化* transiton 1s very intense at190m Peptide bond n-n* transition is weak as it is symmetry forbidden it forms a shoulder on the一>几* transition(emax" 100)at 210-220 nm Aromatic Amino acids-- most useful Phe 8250=400 symmetry forbidden T->T* Tyr274=14007->兀* Trp 280=4500 at least 3 different transitions. A280 is one of the most commonly used methods to measure protein concentration(aside from colorimetric methods such as the Lowry or Bradford dye binding assays), but this method is obviously very sensitive to differences in amino acid composition These transitions can change with pH; especially Tyr Prosthetic Groups Nucleotides ->e.g FMN, NAD Retinal etc

Protein absorbances come from 3 sources Peptide Bond Aromatic Amino Acids -- most useful Phe e250 = 400 symmetry forbidden p --> p* Tyr e274 = 1400 p --> p* Trp e280 = 4500 at least 3 different transitions. A280 is one of the most commonly used methods to measure protein concentration (aside from colorimetric methods such as the Lowry or Bradford dye binding assays), but this method is obviously very sensitive to differences in amino acid composition. These transitions can change with pH; especially Tyr Prosthetic Groups Nucleotides --> e.g. FMN, NAD Heme Cu Retinal etc

Estimating Protein Concentration 1. Generally measure A,0 and assume an average composition of Tyr and Trp: Approximately 1 mg/ml --->1.0A 2. Measure absorbance of peptide groups at2=230 nm 1mg/ml-->3.0A This is not commonly used because many other groups absorb in this wavelength region

Estimating Protein Concentration 1. Generally measure A280 and assume an average composition of Tyr and Trp: Approximately 1 mg/ml ---> 1.0 A 2. Measure absorbance of peptide groups at l = 230 nm: 1 mg/ml ---> 3.0 A This is not commonly used because many other groups absorb in this wavelength region