Lecture 12 - CHO Analysis (Phys, Chrom, Enz)

Question 1

what are physical methods of CHO analysis? (4)

Answer 1

• refractometry
• hydrometry
• pycnometry
• plolarimetry

Question 2

what are instrumental methods of CHO analysis? (4)

Answer 2

• chromatography
• NMR
• Electrophoresis
• IR

Question 3

describe the use of refractometry

Answer 3

• 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

Question 4

where can refractometers be used?

Answer 4

• on processing line

- lab bench

Question 5

what kind of products are commonly assessed with a refractometer?

Answer 5

• carbonated soft drinks
• orange juice (dissolved solids)
• milke (to assess % solids)

Question 6

how is hydrometry used?

Answer 6

• based on Archimedes principle:

- object displaces weight of liquid = own weight

Question 7

what kinds of hydrometers exist?

Answer 7

• lactometer (milk density)
• Baume (salt)
• Brix (saccharometer)
• alcoholometer

Question 8

which hydrometer measures sugar (sucrose) content?

Answer 8

brix hydrometer

Question 9

one degree brix equates to how many grams of sucrose per grams of solution?

Answer 9

1 g sucrose/100 g solution

or % sucrose

Question 10

how is pycnometry used?

Answer 10

based on measuring specific gravity

Question 11

list the 3 steps of pyncometry

Answer 11

1) weigh empty pycnometer
2) fill with water and reweigh
3) fill with sample liquid

Question 12

how is polarimetry used?

Answer 12

• 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

Question 13

what’s it called when light is rotate in a clockwise direction when viewed through a polarimeter?

Answer 13

dextrorotatory

Question 14

what’s it called when plane polarized light is rotated in a counterclockwise direction and viewed through a polarimeter?

Answer 14

levorotatory

Question 15

what is specific rotation?

Answer 15

angle of rotation of plane polarized light by 1.00 gram per cm^-3 sample in a 1 dm tube

Question 16

what is rotation related to?

Answer 16

molecules in solution (aka concentration in g solute/ 1 cc solution)

Question 17

what methods can be used for the specific analysis of mono and oligosaccharides?

Answer 17

• enzymatic methods

- chromatographic methods (HPLC, GC)

Question 18

what’s the name of the treatment used to prepare samples for enzymatic analysis?

Answer 18

Carrez

Question 19

what are the steps of the Carrez treatment?

Answer 19

1) add potassium ferrocyanide
2) add zinc sulfate
3) add NaOH
4) filter suspension
5) use clear suspension for enzyme-catalyzed assays

Question 20

what does the Carrez treatment accomplish?

Answer 20

breaks emulsions, precipitates proteins, & absorbs colors from food products prior to enzymatic determination of CHO

Question 21

how can glucose be analyzed?

Answer 21

Glucose oxidase/peroxidase/dye (GOPOD) method

Question 22

how does the GOPOD method work?

Answer 22

• 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

Question 23

what’s an example of a dye used for the GOPOD method?

Answer 23

4 aminoantipyrine. produces red quinoneimine

Question 24

how do you analyze for starch?

Answer 24

• 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

Question 25

true or false: enzyme purity does not have significant influence on results

Answer 25

false - purity of enzyme is critical

Question 26

presence of which enzymes can lead to overestimation for starch determination?

Answer 26

ones that release glucose not from starch

• cellulase
• invertase
• sucrase
• B-glucanase

Question 27

presence of which enzyme can lead to underestimation for starch determination?

Answer 27

• catalase (destroys H2O2)

Question 28

what kind of starch can’t be detected by enzymatic methods?

Answer 28

enzyme-resistant starch

Question 29

pros of enzymatic methods? (6)

Answer 29

• 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

Question 30

cons of enzymatic methods?

Answer 30

• need clear solution
• must maintain optimal conditions (buffer, pH, temp, etc)
• possible interference from other components

Question 31

how is HPLC used?

Answer 31

• pumps sample mixture in a solvent at high pressure through a column with chromatographic packing material
• column: anion exchanger (AE)
• detector: electrochemical detector (ECD)

Question 32

what is AE-HPLC used for?

Answer 32

detection and quantification of CHO from complex mixtures

Question 33

identification of the CHO is _____ analysis

Answer 33

qualitative

Question 34

how do you perform quantitative analysis using HPLC?

Answer 34

peak integration

Question 35

pros of HPLC?

Answer 35

• relatively fast
• can undertake a wide range of sample concentrations
• high degree of precision and accuracy

Question 36

in what form should sugars be for GC?

Answer 36

derivatized (volatile derivatives)

Question 37

how do you turn sugars into volatile derivatives?

Answer 37

• reduce aldehyde groups to primary OH groups

- convert reduced sugars to volatile peracetate esters

Question 38

what kind of detectors are used for GC?

Answer 38

• flame ionization detector (FID)

- mass spec (MS)