Lecture 12 - Carbohydrates 12 Flashcards
what are 4 physical methods
refractometry
hydrometery
pycnometery
polarimetry
what is refractometry
based on refractive index
absolute refractive index of a medium is the ratio of the speed of light in vacuum relative to the speed in the medium
when light is passed from one medium to another, the density of the 2 differs, then the beam of light is bent or refracted
what is snell’s law
n1sin01 = n2sin02
what law is the principle of refractometry based on?
snell’s law
what does snell’s law state?
that the ratio of the sines of the angles of incidence and refraction is equivalent to the ratio of phase velocities in the 2 media
what is a characteristic property of a liquid?
the refractive index
what is the refractive index of a solution related to?
density of the solution
what does the refractive index depend on?
concentration of the solution
temp
wavelength of light
what is the refractive index of pure water?
what happens when sugar is added?
n20 = 1.33299
sugar changes density of water. Leads to change in refractive index
what can refractometers used for?
liquid processing to monitor products such as carbonated soft drinks, dissolved solids in orange juice, %solids in milk
what are 2 types of refractometers?
Abbe refractometer: handheld and benchtop model. Has wider measurement range and more precision. LEss portability
digital refractometer
what is the principle behind hydrometry
based on Archimede’s principle: an object displaces a weight of liquid equals to its own weight
thus it is based on displacement of a liquid by a floating body
how do you calculated the weight of an object (in hydrometry)
calculate weight of the displaced liquid
wt = volume x density
the weight of the displaced liquid is equal to what?
wt of the object
what is the equation of sepcific gravity?
SG = density of liquid / density of water
what are the 4 types of hydrometers?
- lactometer: determines density of milk
- Baume: used to determine density of salt
- Brix: (saccharometer) used for sugar solutions
- alcoholmeter: used to estimate alcohol content of beverages
brix is a wt measurement system calibrated using what?
sucrose solution concentration
one degree brix = ____ g of sucrose in ___ grams of solution?
brix = ____
20 brix = _____
1 gram sucrose in 100 grams solution
brix = % sucrose
20 brix = 20% sucrose
what is the basis of pycnometery?
based on measuring SG
density equation?
d=w/v
what are the steps in pycnometery?
- weigh empty pycnometer (W1)
- fill it with water and reweigh (W2)
- fill it with liquid (W3)
SG = W3-W1 / W2-W1
what is the SG of a 0% sugar solution concentration?
1.00
what is the SG of a 10% sugar solution concentration?
1.0320
what molecules can rotate plane polarized light?
molecules with an asymmetric C atom (chiral molecules)
optical active substances
sugars are optically active because….
they are chiral
what does chiral mean?
that a component is not superimposable on the mirror image
chiral molecules are optically active
what substances are optically active?
substances that can change the plane of polarized light
eg proteins, organic acids, AAs, sugars
what is a polarimeter?
an instrument used to measure optical activity
how do polarizers work?
what are the inputs and outputs?
they absorb one component of the polarization but not the other
input: natural light
output: polarized light (linear, circular, elliptical)
they work by dichroism, reflection or scattering
what is dextrorotary
when the plane of polarized light is rotated in a clockwise direction when viewed through a polarimeter
(+) of (d)
do not confuse with D
what is levorotary
when the plane of polarized light is rotated in a counter clockwise direction when viewed thorugh a polarimeter
(-) or (l)
do not confuse with L
the angle of rotation of plane polarized light by an optically active substance is porportional to what?
the number of atoms in the path of hte light
what is specific rotation?
what is the equation?
the angle of rotation of plane polarized light by a 1 gram per cm sample in a 1dm tube
[alpha] = alpha / l * d
where alpha = observed rotation
l = length (dm)
d = concentration (g/cc)
what is polarimetry used for?
what is it not good for?
determining the purity of sugar solutions
not good for unknown mix
what are 2 methods of mono and oligosaccharide analysis?
- enzymatic methods
2. chromatographic methods: high performance liquid chromatography and gas chromatography
what are 2 types of chromatography methods
high performance liquid chromatography and gas chromatography
what is high performance liquid chromatography? what are advantages?
what type of analysis does it require?
- selction of mobile phase column (anion exchange) and dector (pulsed electrochemical detector
- couple with an ECD for detection and quantification of CHO from complex minutes
- involves: qualitative analysis (ID of the CHO) and quantitative analysis (peak integration)
adv:
- relatively faster
- possibility of wide range of sample conc
- high degree of precision and acuracy
what is gas chromatography?
sugars are converted into what? what reaction occurs?
what detector is used?
- qualitative and quantitate analysis of CHO
- sugars are converted into volatile derivatives
- reduction of adehydic groups to primary OH groups and conversion of the reduced sugars (alditol) into volatile peracetate esters
detector: flame ionization detector (FID) or mass spectrometric (MS)
what is FID
flame ionization detector
what is MS
mass spectrometric
what are advantages of enzymatic methods?
disadvantages?
adv:
- higher specificity
- does not require high purity of sample being analyzed
- has low detection limits
- does not require expensive equipment
- can be easily automated
- commercially available kits
dis:
- need clear solution
- optimum condition: buffer, pH, temp
- possible interference from other components
how is the sample prepared in enzymatic methods?
what treatment is used?
- carrez treatment: breaks emulsions, precipitates, proteins, absorbs colors from food products prior to enzymatic determination of CHO
- add potassium ferrocyanid
- addition of zinc sulfate (ZnSO4)
- addition of sodium hydroxide
- filtration of suspension
- use the clear suspension for enzyme-catalyzed assays
what is the GOPOD method
glucose oxidase/peroxidase/dye method
- glucose oxidase oxidized d-glucose quantitatively to d-glucono-1,5-lactone (glucono-delta-lactone) and hydrogen peroxide
- D-glucose is measured from H2O2 generated using peroxidase that turns a colorless compound (leuco dye) to a colored compound
- measure colored compound spectrophotometrically
describe the enzymatic method of starch
what are the important enzymes
- starch is gelatinized in hot DMSO
- starch solution is digested with alpha-amylase
- linear and branched fragments of amylose and amylopectin are digested with glucoamylase
- d-glucose is reacted with the GOPOD reagent
- color solution
what is alpha-amylase?
1,4-linked alpha-d-glucopyranosyl units
what are important enzymes in starch enzymatic analysis?
- alpha-amylase
2. glucoamylase
what reagent is used in starch enzymatic analysis?
GOPOD reagent
in the starch enzymatic method, what results in a false high value? and a false low value?
false high value: when cellulases, invertase, sucrase and beta-glucanase releases D-glucose from CHO other than starch
false low value: catalase destroys the H2O2
in the starch enzymatic method, what is not detected?
enzyme resistant starch
what is critical in the starch enzymatic method?
purity of enzyme used
