Analysis of Carbohydrates - Detectros
10 important questions on Analysis of Carbohydrates - Detectros
Why are UV detectors not used for carbohydrate detection?
How is the refractive index (RI) used?
- Two cells present: reference and sample cell
- Reference cell filled with eluent without any compound
- Quite sensitive to environmental conditions (T, buffer, impurities)
- Universal detection / quantification
- No gradient elution allowed
How does multi angle light laser scattering work?
- Reflection of light by molecules/ particles measured at different angles
- Accurate measurement of Mw of molecule or aggregate
- Information on molecules shape and flexibility
- Discriminative towards small molecules
- Over-estimating aggregates and high Mw populations
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How does viscometric detection work?
- Pressure over different coils is measured and correlated to the viscosity
- Discriminative towards small molecules
- Keeping a constant temperature is very important.
How does a evaporative light scattering detector (ELSD) work?
- ELSD is prefered over RI since you can use a elution gradient because the eluent is evaporated.
- Non linear response for OS above DP4
How does the Pulsed Amperometric Detector (PAD) work?
- PAD gives different responses for different sugars.
- Lower for monosaccharides
How does laser-induced fluorescence detection (LIF) work?
- Molecules are first excited to higher energy levels by laser light and then emit light of lower wave lengths.
- Carbohydrates have to be labeled with APTS since they are not fluorescent of them selves.
- Good in combination with MS in order to identify carbohydrates.
Why can you analyse arabinose oligomers easyere with PGC-HPLC then with HPAEC-PAD?
Why is there a difference in response and/or shape of peaks and the different elution time(s)?
LS: Firs half gives more signal because it positively discriminates the big molecules, underestimates the intermediate ones and overlooks the small ones
IV: You don't see the small molecules, discrimination is based on MW.
Why are at least two detectors needed for a good evaluation? Which ones?
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