FOOD ANALYSIS: Carbohydrate Analysis
B. Pam Ismail [email protected] FScN 146 612 625 0147
FOOD ANALYSIS: Carbohydrate Analysis
The following is/are a carbohydrate(s):
A. Pectin B. Cellulose C. Lignin D. A & B E. B & C F. A & C G. All of the above H. None of the above
1 FOOD ANALYSIS: Carbohydrate Analysis
The following is/are a carbohydrate(s):
A. Pectin B. Cellulose C. Lignin D. A & B E. B & C F. A & C G. All of the above H. None of the above
FOOD ANALYSIS: Carbohydrate Analysis
The method used to determine starch gelatinization could be used to determine starch retrogradation
A. True B. False
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Importance of Carbohydrates Importance of Analyzing Carbohydrates
FOOD ANALYSIS: Carbohydrate Analysis
Carbohydrate Classification
CH2OH CH2OH HO O O OH OH OH Monosaccharides O
Di and oligosaccharides (2-10 units) OH OH
Polysaccharides CH2OH HOH C O O 2 OH o Starch HO O HO CH2OH o Dietary fiber OH OH
CH2OH CH2OH O O OH OH
O O O
OH OH CH2OH CH2OH CH2OH CH2 CH2OH CH2OH O O O O OH OH O O OH OH OH OH O O O O O O O OH OH OH OH OH OH
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General Sample Preparation
Drying o Vacuum oven
Fat extraction o Soxhlet
FOOD ANALYSIS: Carbohydrate Analysis
Total Carbohydrate Analysis H (research purposes) HO O Phenol-sulfuric acid method O What happens to glycosidic linkages under acidic conditions? strongly acidic conditions furans monosaccharides (heat)
CH2OH CH2OH O O enolizations, OH OH OH dehydrating reactions H O HO O OH OH O
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Total Carbohydrate Analysis (research purposes) Phenol-sulfuric acid method Furfural derivatives (furans) react with phenols to produce colored compounds (triarylmethane dyes) Absorption maxima between 480 and 490 nm
OH
OH absorption @ 490 nm concentration + H2SO4 2 O R OHC O R
OH Absorption @ 490 nm 490 @ Absorption Not a Stoichiometric reaction concentration
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Somogyi-Nelson Colorimetric Munson-Walker Based on reducing Methods Cu+2 to Cu+1 Lane-Eynon
Paper and TLC Chromatographic Methods HPLC
GC
Enzymatic Methods
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Extraction
Solvent extraction o mono- and disaccharides are soluble in water and also in 80% ethanol o most polysaccharides and proteins are insoluble in 80% ethanol o mono/disaccharide extraction using hot 80% ethanol o neutral pH conditions required to prevent hydrolysis
Carrez treatment o clarification with Carrez-reagents
o Potassium hexacyanoferrate K4[Fe(CN)6] solution, ZnSO4 o Breaks emulsions o precipitates proteins and partially polysaccharides o absorbs colors
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Colorimetric For reducing sugars Methods
Principle: Based on reduction of cupric ions in alkaline solution to cuprous ions by reducing sugars; reaction with a complex compound to generate color; amount of cuprous ions generated is measured to quantitate reducing sugars (gravimetric or titration)
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Mono- and Oligosaccharide Analysis
Colorimetric For reducing sugars Methods
Principle: Based on reduction of cupric ions in alkaline solution to cuprous ions by reducing sugars; reaction with a complex compound to generate color; amount of cuprous ions generated is measured to quantitate reducing sugars (gravimetric or titration)
CHO COOH H OH Cu2+ H OH HO H Cu2O + HO H - H OH OH H OH
H OH H OH CH OH 2 CH2OH What is wrong?
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Colorimetric For reducing sugars Methods
Lane-Eynon Somogyi-Nelson Munson-Walker
Cu(II) complexes and Quantification: reagents uses: -Determination of Cu2O by -Citrate gravimetric procedures or titrimetric procedures (sodium - ethylenediaminetetraacetic Differences in Methods acid (EDTA) thiosulfate or potassium permanganate - tartrate - spectrophotometric assays - pH and reagent to create (reduction of arsenomolybdate, alkaline conditions (NaOH, blue color – 520 nm) KOH, Na2CO3)
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Mono- and Oligosaccharide Analysis
Colorimetric For reducing sugars Methods
What carbohydrates do you analyze using the Somogyi-Nelson method and other reduction methods?
How can you analyze sucrose?
CH2OH HOH C O O 2 OH HO O HO CH2OH OH OH
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Very popular approach in the field of carbohydrate Enzymatic analysis Methods Enzymes are proteins with powerful catalytic activity (lower the activation energy) High specificity for both the compound to be converted and for the type of reaction
enzyme
reactant
enzyme
product
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Mono- and Oligosaccharide Analysis
Enzymatic food analysis involves determination of food Enzymatic constituents which are usually substrates for enzymes Since the final determination is a spectrophotometric Methods assay solutions have to be clarified, sometimes enzyme- inhibitors have to be removed Very often two types of reaction are necessary since it is not possible to measure the reactant or the product
Measuring the reaction food Auxiliary reaction constituent A + B P + Q
Indicator reaction P + C R + S
C, R or S
FOOD ANALYSIS: Carbohydrate Analysis
glucose Auxiliary reaction A + B P + Q
D-Glucose + ATP Hexokinase Glucose-6-phosphate + ADP
Indicator reaction NADPH P + C R + S
Glucose-6-phosphate + NADP+ G6P-DH 6-Phosphogluconate + NADPH + H+
G6P-DH - Glucose-6-phosphate-Dehydrogenase
9 FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Enzymatic Methods Example Applications:
o Monosaccharides Fructose, Glucose, Galactose
o Oligosaccharides Lactose, Maltose, Sucrose
o Polysaccharides Amylose, Amylopectin
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Enzymatic Methods Advantages:
Usually specific for substance being measured Kits commercially available Generally have low detection limits
Disadvantages:
Interfering compounds maybe present Not always 100% specific
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Mono- and Oligosaccharide Analysis
Chromatographic Paper and TLC Methods mostly qualitative information, however, TLC also used for quantitation
Visualized by autoradiography
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Chromatographic Paper and TLC Methods
TLC: stationary phases: cellulose, silica or impregnated silica mobile phase, e.g.: similar to paper Paper chromatography: Chromatography; 1-butanol-acetone-H2O stationary phase: cellulose filter; 4:5:1 (v/v); 1-butanol-ethanol-H2O mobile phase: e.g. n-propanol/ethyl 2:1:2 (v/v) (for acetate impregnated silica) acetate/H2O (65/10/25 v/v/v); Silica impregnation: acetates (0.02 – 0.2 M) or phosphates (0.07 – 0.5M) improved separation/resolution, spots are sharpened
11 FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Chromatographic HPLC Methods
Anion-Exchange HPLC Normal-Phase HPLC Cation-Exchange HPLC Reversed-Phase HPLC
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Normal-Phase HPLC
Stationary phase: unmodified silica
strong interactions between free silanol groups of the silica gel and the hydroxyl groups of the carbohydrates
good separation of carbohydrates almost impossible
Use chemically modified silica stationary phases! Also called HILIC (Hydrophilic Interaction Liquid Chromatography)
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Mono- and Oligosaccharide Analysis
Normal-Phase HPLC
Chemically modified silica stationary phases (HILIC):
amine-bonded stationary phases (e.g. -C3H6NH2)
mobile phase usually acetonitrile/water (50-85% acetonitrile)
separation may be influenced by the column temperature
formation of hydrogen bonds between amino groups and hydroxyl groups
FOOD ANALYSIS: Carbohydrate Analysis
Recap Questions
How do we measure total carbohydrates?
What are the preparatory steps for carbohydrate analysis?
How do we extract mono, di and oligosaccharides?
Name methods used to determine mono, di, and oligosaccharides
What is the basis of carbohydrate analysis using enzymes?
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Mono- and Oligosaccharide Analysis Normal-Phase HPLC ?
Amine-bonded stationary phases
Detection: usually RI-detection
Drawbacks: gradient elution is not possible, insensitive and highly unspecific
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Normal-Phase HPLC
Drawbacks of amine-bonded stationary phases: H+ O H OH H limited column life due to the formation C H+ of Schiff bases between the amino H C OH H C NR HNRC R-NH2 group and the sugar molecules HO C H H C OH H C OH -H O H C OH HO C H 2 HO C H H C OH H C OH H C OH Modifiers CH2OH H C OH H C OH CH2OH CH OH Glucoseglucose 2 ImineImin (Schiff Other modifications available for uses in carbohydrate analysis: base) … diol-bonded (e.g., -C3H6OC2H3OHCH2OH) stationary phases
cyano-bonded stationary phases (e.g., -C2H4CN)
14 FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Reversed-Phase HPLC
Stationary phase: e.g. RP-18-columns
Used for the analysis of gluco-oligomers with water as mobile phase
Separation of different monosaccharides is difficult….Why? What can be done?
Reversed-phase chromatography is usually affected by anomers, resulting in peak doubling or broadening (use amine groups in mobile phase to acceleraate anomerization)
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Anion-Exchange HPLC
In solution of high pH carbohydrate hydroxyl groups are ionized (weak acids),
Polystyrene-divinylbenzene copolymers are often used
Detection is mostly by pulsed amperometric detection (PAD)…pulsed electrochemical detector (ECD)
Order of elution??
15 FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Anion-Exchange HPLC
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Cation-Exchange HPLC
Most commonly applied: cation exchange resins are porous polystyrene-divinylbenzene matrices
Loading of sulfonated ion exchangers with inorganic counter ions (Ca2+, Ag+) affects the retention of non- ionized carbohydrates Counter ion: Metal counter ions are able to bind sample calcium molecules through coordination complexes
o Ion exchange o Size exclusion
Mobile phase: water for resins using metals as counter ions, diluted acids using protons as counter- ions
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Mono- and Oligosaccharide Analysis
Chromatographic Gas Chromatography Methods
OH How can we analyze O OH OH carbohydrates by GC?
HO OH
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Gas Chromatography Silylated derivatives more volatile
OH than acetylated derivatives
O OH OH Silylation suitable for disaccharides
HO OH Acetylation only for monosaccharide Derivatization: silylation, acetylation, trifluoroacetylation
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Mono- and Oligosaccharide Analysis
Gas Chromatography O H H C HOHC H C OH H C OH HO C H Reduction to the sugar alcohols HO C H H C OH NaBH 4 H C OH H C OH H C OH CH2OH CH2OH
…followed by e.g. acetylation
H H HOHC HOAcC H C OH H C OAc HO C H AcO C H H C OH H C OAc Acetic Anhydride H C OH H C OAc CH OH 2 CH2OAc
FOOD ANALYSIS: Carbohydrate Analysis
Mono- and Oligosaccharide Analysis
Gas Chromatography
Erythritol Inositol GC-FID Man Glc chromatogram of Ara Fu Xyl Gal neutral Rhac monosaccharides Column: HP-5MS- Fused-Silica- capillary columns, Injection: on- column; Detector: FID, 300 °C; carrier gas: He flow rate: 1.5 mL/min
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Polysaccharide Analysis
Total Starch
Starch Degree of Gelatinization
Degree of Retrogradation
Pectin Dietary Fiber Total Dietary Fiber
FOOD ANALYSIS: Carbohydrate Analysis
Total Starch
Qualitative Analysis: Amylose has an affinity for iodine; enclosed by the starch, iodine exhibits a strong absorption of light (intense blue color)
Microscopy
Quantitative Analysis: rye barley oats Enzymatic methods
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Total Starch
Quantitative Analysis: ? Enzymatic methods: Starch is converted totally into D-glucose by enzymes specific for starch, and D-glucose is quantitated enzymatically
-gluconolactone
peroxidase H2O2 + ο-diansidine H2O + oxidized dye (colored)
FOOD ANALYSIS: Carbohydrate Analysis
Amylases
glucoamylase endo- enzyme exo- enzymes pullulanase
β-amylase
non-reducing end reducing end
α-amylase
endo- Amylose vs. Amylopectin enzyme
20 FOOD ANALYSIS: Carbohydrate Analysis
Total Starch
Quantitative Analysis: ? Enzymatic methods: Starch is converted totally into D-glucose by enzymes specific for starch, and D-glucose is quantitated enzymatically
-gluconolactone
peroxidase H2O2 + ο-diansidine H2O + oxidized dye (colored)
FOOD ANALYSIS: Carbohydrate Analysis
Degree of Gelatinization & Degree of Retrogradation
Employing a combination of pullanase and β-amylase
De-branches
A mixture of linear segments of various sizes Maltoses and maltotrioses
Measure amount of reducing sugars formed
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Dietary Fiber
Dietary fiber is the edible part of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine Dietary fiber includes polysaccharides, oligosaccharides (oligofructans), lignin
Soluble fiber: Pectin and hydrocolloids/gums. Beans, oat bran, fruit and vegetables contain soluble fiber Insoluble fiber: fibers in cell walls, cellulose, hemicellulose, lignin and resistant starch. Wheat bran and whole grains contain the most insoluble fiber Is it possible to analyze all dietary fiber compounds in a single approach?
FOOD ANALYSIS: Carbohydrate Analysis
Dietary Fiber
Crude fiber vs. total Dietary fiber
Crude fiber
Acid and alkaline series of extractions
Detergent extraction (animal feed industry)
Not sufficient for human food analysis
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Total, insoluble and soluble fiber (AOAC method 991.43) (Pre-dried and fat free Sample)
Digest with -amylase (a) Adjust pH w/ buffer Control pH, Heat sample to Digest with protease (b) times, temp. gelatinize starch Digest with glucoamylase (c) Ppt. w/ ethanol; Wash; Dry; Filter and wash Correct for (d) protein & ash
TDF
FOOD ANALYSIS: Carbohydrate Analysis
Total, insoluble and soluble fiber (AOAC method 991.43)
Residue (Insoluble fiber)
Wash with water, 95% ethanol, and acetone (e)
Oven dry (f)
Weigh (g)
Determine residual protein & ash (h) Calculate insoluble fiber
23 FOOD ANALYSIS: Carbohydrate Analysis Total, insoluble and soluble fiber (AOAC method 991.43) Filtrate and washings (Soluble fiber) Precipitate with ethanol and filter (j)
Precipitate Solubles (Soluble fiber) Wash with 78% ethanol (k) Wash with 95% ethanol and acetone (l) Oven dry (m) Weigh (n)
Determine residual protein (o) Ash (p) Calculate soluble fiber
FOOD ANALYSIS: Carbohydrate Analysis
Total, insoluble and soluble fiber (Englyst-Cummings method)
Starch is gelatinized and digested by enzymes Protein is hydrolyzed by proteases Remaining non-starch polysaccharides are hydrolyzed by sulfuric acid to liberate free monosaccharides
o Neutral sugars--- GC-FID o Uronic acids by colorimetric detection
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Pectin Analysis
Precipitation with alcohol (jams, jellies etc.)
Units of galacturonic acid---glycosidic linkages of uronic acids are hard to hydrolyze with acid
Saponification with NaOH followed by acidification and ppt with Ca+2----Calcium pectate (gravimetric method)
FOOD ANALYSIS: Carbohydrate Analysis
The following data were obtained when a high-fiber cookie was analyzed for fiber content by the AOAC Method 991.43 Sample wt. 1.0021 1.0053 Sample (g)
(g) Insoluble Soluble
Crucible 31.6372 32.1739 32.3775 33.2164 Blank Crucible + 31.7235 32.2712 32.4216 33.2553 Residue Insoluble Soluble Protein 0.0065 0.0039 Crucible 31.5636 32.1987 33.0196 33.9812 Crucible + 32.1952 33.2310 Crucible + 31.5782 32.2132 33.0334 33.9956 Ash Residue Protein 0.0032 0.0033 Calculate % total, insoluble and soluble Fiber Crucible + 32.2068 33.9891 Ash
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Av sample wt =
Insoluble R1 and R2, get the Av for sample and for blank (subtract value of blank from value of sample)
Subtract blank protein from protein of insoluble residue
Subtract blank ash from ash of insoluble residue
(Av insoluble R – protein –ash / Av wt of sample) x 100
Repeat the same of the soluble
% soluble + % insoluble = Total fiber
FOOD ANALYSIS: Carbohydrate Analysis
Total Carbohydrate Analysis
Determine total carbohydrate in beverages following Phenol- sulfuric acid method
Read Chapter 19 (text book) and Chapter 14 (lab manual) found in your manual
Complete pre-lab quiz
Bring Lab manual and laptops
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