FOOD ANALYSIS: Analysis

B. Pam Ismail [email protected] FScN 146 612 625 0147

FOOD ANALYSIS: Carbohydrate Analysis

 The following is/are a carbohydrate(s):

A. B. C. 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 gelatinization could be used to determine starch retrogradation

A. True B. False

2 FOOD ANALYSIS: Carbohydrate Analysis

 Importance of  Importance of Analyzing Carbohydrates

FOOD ANALYSIS: Carbohydrate Analysis

 Carbohydrate Classification

CH2OH CH2OH HO O O OH OH OH  O

Di and oligosaccharides (2-10 units) OH OH

CH2OH HOH C O O 2 OH o Starch HO O HO CH2OH o 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

3 FOOD ANALYSIS: Carbohydrate Analysis

 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

4 FOOD ANALYSIS: Carbohydrate Analysis

 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

5 FOOD ANALYSIS: Carbohydrate Analysis

 Extraction

Solvent extraction o mono- and are soluble in water and also in 80% ethanol o most polysaccharides and are insoluble in 80% ethanol o mono/ extraction using hot 80% ethanol o neutral pH conditions required to prevent

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 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)

6 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)

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)

7 FOOD ANALYSIS: Carbohydrate Analysis

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 ?

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

8 FOOD ANALYSIS: Carbohydrate Analysis

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

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 , Glucose,

o Oligosaccharides , , Sucrose

o Polysaccharides ,

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

10 FOOD ANALYSIS: Carbohydrate Analysis

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)

12 FOOD ANALYSIS: Carbohydrate Analysis

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?

13 FOOD ANALYSIS: Carbohydrate Analysis

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 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- with water as mobile phase

 Separation of different monosaccharides is difficult….Why? What can be done?

 Reversed-phase chromatography is usually affected by , 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

16 FOOD ANALYSIS: Carbohydrate Analysis

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 Derivatization: silylation, acetylation, trifluoroacetylation

17 FOOD ANALYSIS: Carbohydrate Analysis

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

18 FOOD ANALYSIS: Carbohydrate Analysis

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

19 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

 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

21 FOOD ANALYSIS: Carbohydrate Analysis

Dietary Fiber

 Dietary fiber is the edible part of plants or analogous carbohydrates that are resistant to and absorption in the human with complete or partial fermentation in the  Dietary fiber includes polysaccharides, oligosaccharides (oligofructans), lignin

 Soluble fiber: Pectin and hydrocolloids/gums. , oat bran, fruit and vegetables contain soluble fiber  Insoluble fiber: fibers in cell walls, cellulose, , lignin and . 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

22 FOOD ANALYSIS: Carbohydrate Analysis

 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 (b) times, temp. gelatinize starch Digest with glucoamylase (c) Ppt. w/ ethanol; Wash; Dry; Filter and wash Correct for (d) & 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  Remaining non-starch polysaccharides are hydrolyzed by sulfuric acid to liberate free monosaccharides

o Neutral sugars--- GC-FID o Uronic acids by colorimetric detection

24 FOOD ANALYSIS: Carbohydrate Analysis

 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

25 FOOD ANALYSIS: Carbohydrate Analysis

 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

26