Analysis of Carbohydrates - Edubirdie

University:
Swansea University
Course:
PM134 | Microbiology
Academic year:

2023
Views:

434

Pages:

3
Author:

Amal J.

How to analyze carbohydrates: Determine if it's a reducing or non-reducing sugar. Classification: Endo or exo enzymes. Amylase and Starch: For amylase tests, determine what is used as a standard. Common β-amylase has an end product of maltose. Depending on the pH, frame the question/experiment. Using enzymes to determine concentration via UV absorption: Enzymatic oxidation of carbohydrates can be measured using UV absorption or colorimetric methods. Example Calculation: NADPH Concentration Calculation: Molar extinction coefficient ( 𝜖 ϵ) = 5000 M − 1 −1 cm − 1 −1 𝐴 = 𝜖 𝑐 𝑙 A=ϵcl Given: 𝐴 = 0.8 A=0.8, 𝑙 = 1 l=1 cm 𝑐 = 0.8 5000 c= 5000 0.8 Tryptophan Absorbance: Wavelength: 280 nm 𝐴 = 𝜖 𝑐 𝑙 A=ϵcl Given: 𝐴 = 0.54 A=0.54, 𝑙 = 0.5 l=0.5 cm, 𝜖 = 6.4 × 1 0 3 ϵ=6.4×10 3 L mol − 1 −1 cm − 1 −1 𝑐 = 0.54 ( 6.4 × 1 0 3 ) × 0.5 c= (6.4×10 3 )×0.5 0.54 Calculating Concentration: Given thickness 𝑙 = 2 l=2 cm Incident light wavelength: 410 nm 𝐴 = log ⁡ 𝑇 A=logT 𝜖 = 6000 ϵ=6000 dm 3 3 mol − 1 −1 cm − 1 −1 𝑇 = 1 0 − 𝐴 T=10 −A Find the concentration using 𝐴 = 𝜖 𝑐 𝑙 A=ϵcl. Frequency Calculation: Frequency 𝜈 ν = 𝑐 𝜆 λ c Given 𝜈 = 4.25 × 1 0 14 ν=4.25×10 14 Hz Find wavelength 𝜆 λ. Questions: How to quantify a protein using UV-Vis? Separate UV and Vis ranges. NADH and NAD: Different absorbance properties. KBr and NaCl: Do not absorb IR. Coenzymes absorbance: FAD and FADH.

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