Lecture 12 - CHO Analysis (Phys, Chrom, Enz) Flashcards
what are physical methods of CHO analysis? (4)
- refractometry
- hydrometry
- pycnometry
- plolarimetry
what are instrumental methods of CHO analysis? (4)
- chromatography
- NMR
- Electrophoresis
- IR
describe the use of refractometry
- based on refractive index
- relies on Snell’s law (n1sina=n2sinb)
- refractive index is related to density of solution
- depends on concentration, temperature, and wavelength
- inc of [sugar] changes water density, and therefore a increases refractive index
where can refractometers be used?
- on processing line
- lab bench
what kind of products are commonly assessed with a refractometer?
- carbonated soft drinks
- orange juice (dissolved solids)
- milke (to assess % solids)
how is hydrometry used?
- based on Archimedes principle:
- object displaces weight of liquid = own weight
what kinds of hydrometers exist?
- lactometer (milk density)
- Baume (salt)
- Brix (saccharometer)
- alcoholometer
which hydrometer measures sugar (sucrose) content?
brix hydrometer
one degree brix equates to how many grams of sucrose per grams of solution?
1 g sucrose/100 g solution
or % sucrose
how is pycnometry used?
based on measuring specific gravity
list the 3 steps of pyncometry
1) weigh empty pycnometer
2) fill with water and reweigh
3) fill with sample liquid
how is polarimetry used?
- molecules (like sugars) w/asymmetric C atom can rotate plane polarized light
- polarimeter is used to measure optical activity to obtain specific rotation of a solution
what’s it called when light is rotate in a clockwise direction when viewed through a polarimeter?
dextrorotatory
what’s it called when plane polarized light is rotated in a counterclockwise direction and viewed through a polarimeter?
levorotatory
what is specific rotation?
angle of rotation of plane polarized light by 1.00 gram per cm^-3 sample in a 1 dm tube
what is rotation related to?
molecules in solution (aka concentration in g solute/ 1 cc solution)
what methods can be used for the specific analysis of mono and oligosaccharides?
- enzymatic methods
- chromatographic methods (HPLC, GC)
what’s the name of the treatment used to prepare samples for enzymatic analysis?
Carrez
what are the steps of the Carrez treatment?
1) add potassium ferrocyanide
2) add zinc sulfate
3) add NaOH
4) filter suspension
5) use clear suspension for enzyme-catalyzed assays
what does the Carrez treatment accomplish?
breaks emulsions, precipitates proteins, & absorbs colors from food products prior to enzymatic determination of CHO
how can glucose be analyzed?
Glucose oxidase/peroxidase/dye (GOPOD) method
how does the GOPOD method work?
- Glucose oxidase oxidizes d-glucose to d-glucono-1,5-lactone and hydrogen peroxide
- peroxidase is added to react with H2O2, turning a colorless dye into a colored compound
- colored compound measured w/spec
what’s an example of a dye used for the GOPOD method?
4 aminoantipyrine. produces red quinoneimine
how do you analyze for starch?
- prepare sample by first gelatinizing in hot DMSO
- digest result with a-amylase (makes linear & branched fragments of amylose and amylopectin)
- digest this with glucoamylase (makes glucose)
- use GOPOD reagent on d-glucose (makes color)
- measure with spec
true or false: enzyme purity does not have significant influence on results
false - purity of enzyme is critical
presence of which enzymes can lead to overestimation for starch determination?
ones that release glucose not from starch
- cellulase
- invertase
- sucrase
- B-glucanase
presence of which enzyme can lead to underestimation for starch determination?
- catalase (destroys H2O2)
what kind of starch can’t be detected by enzymatic methods?
enzyme-resistant starch
pros of enzymatic methods? (6)
- HIGH specificity
- doesn’t require high purity of sample
- very low detection limits
- do not require expensive equipment
- can be easily automated
- commercially available kits
cons of enzymatic methods?
- need clear solution
- must maintain optimal conditions (buffer, pH, temp, etc)
- possible interference from other components
how is HPLC used?
- pumps sample mixture in a solvent at high pressure through a column with chromatographic packing material
- column: anion exchanger (AE)
- detector: electrochemical detector (ECD)
what is AE-HPLC used for?
detection and quantification of CHO from complex mixtures
identification of the CHO is _____ analysis
qualitative
how do you perform quantitative analysis using HPLC?
peak integration
pros of HPLC?
- relatively fast
- can undertake a wide range of sample concentrations
- high degree of precision and accuracy
in what form should sugars be for GC?
derivatized (volatile derivatives)
how do you turn sugars into volatile derivatives?
- reduce aldehyde groups to primary OH groups
- convert reduced sugars to volatile peracetate esters
what kind of detectors are used for GC?
- flame ionization detector (FID)
- mass spec (MS)
