First HPLC in a Louisiana Factory: What it Can Do for You First HPLC in a Louisiana Factory: What it Can Do for You Gillian Eggleston, David Stewart, Belisario Montes, Lynda Wartelle, and Chardice Verret

 What is HPLC?  What type of factory samples can be analyzed?  What makes up a HPLC system at a factory?  Training for the interpretation of results  What results can do for you - Glucose/fructose ratios - mannitol - sucrose losses  How are factory samples prepared?  Summary: Pros and Cons 2 Presentation Outline

What is HPLC? HPLC = high performance liquid chromatography  HPLC is an analytical chemistry technique to separate, identify, and quantify each component in a mixture Mixture: Sugar factory product, e.g., juice, syrup, molasses Component: sucrose, glucose, fructose, mannitol, short-chain sugars Mixture: Sugar factory product, e.g., juice, syrup, molasses Component: sucrose, glucose, fructose, mannitol, short-chain sugars  HPLC relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with solid absorbent material. Each component interacts slightly differently with the adsorbent material, leading to separation of components as they flow out of the column into a detector Basic HPLC Scheme

What Makes Up a HPLC System HPLC System at Alma Factory (2015) Basic HPLC Scheme detector column pump solvents Auto- sampler data aquisition

What Makes Up a HPLC System HPLC System at Alma Factory (2015) detector column pump solvents auto- sampler data aquisition detector column auto-sampler pump

Data Aquisition

Preparation of Samples Factory Samples Dilutions

Preparation of Samples  Diluted samples must be filtered through 0.45 micron filters to remove suspended samples

Types of Samples Routinely Analyzed

Sample Mannitol Glucose FructoseMannitol Glucose Fructose RS Ratio ID Brixppm %/Brix F/G Crusher 7 am Crusher 11 am 18.30n.d Crusher 1 pm Mixed J Clear Dorr Clear Grav Clear Grav Syrup A Syrup B Final Mol 5 am Final Mol 9 pm Alma Plantation LLC. Lab Report Date: 8 Nov 2015 n.d.=not detected RS=reducing sugars (glucose + fructose)

Types of Samples Routinely Analyzed mixed juice

Types of Samples Routinely Analyzed Syrup Set A; 07:00 am Clear Juice; Graver 2; 24 h composite

Types of Samples Routinely Analyzed Final molasses; 24 h composite

Training

HO O OH OH CH 2 OH OH CH 2 O HO O CH 2 OH protonation of glycosidic oxygen Sucrose CH 2 OH HO O CH 2 OH HO + Fructose Carbocation (Fru + ) O OH OH CH 2 OH OH Glucose* + Fructose* H2OH2O “Acid” (up to pH 8.3) Thermal Degradation or Inversion of Sucrose in Aqueous Solution OH HO O CH 2 OH OH HOCH2  Under both acidic and alkaline conditions, fructose is less stable than glucose: glucose>fructose and the G/F ratio is > 1.0 or the F/G ratio is < 1.0

Effect of Invert Sugars Invert sugars can catalyze the acid degradation of sucrose Then Mechanism Glucose + Fructose acid conditions in juice Acids + color (H + ) compounds O OH OH CH 2 OH OH CH 2 O HO O CH 2 OH Sucrose O OH OH CH 2 OH OH Glucose + Fructose OH HO O CH 2 OH OH HOCH2 H+H+

From: Parker, Cane Sugar Handbook Percentage of Sucrose Acid Degraded in One Hour TempBrix pH o C o C

Training

Is Fructose greater than Glucose? Yes No Is this Juice? Yes Dextran and mannitol have formed No Is this syrup, massecuites, or molasses? Maillard color reactions have occurred Is Glucose greater than Fructose? Yes Are conditions acidic? YesNo Acid degradation of sucrose & Invert has occurred Alkaline degradation of invert has occurred Yes Training

F/G is > 1.0 in upstream crusher and mixed juice = dextran F/G is > 1.0 in downstream molasses and massecuites = Maillard color reactions

Mannitol

Leuconstoc mesenteroides mannitol dehydrogenase Fructose Mannitol + lactic + acetic + CO 2 acid acid n Sucrose G-F Dextran (glucose) n + fructose H2OH2O dextransucrase acceptors isomaltooligosaccharides (up to 7dp) leucrose, palatinose Glucose lactic acid + ethanol + CO 2 lactic acid + acetic acid + CO 2 O2O2 No O 2 phosphoketolase Major Metabolites of Hetero-fermentative Leuconostoc Bacteria of Interest to the Worldwide Sugarcane Industry

Antibody Dextran ASI Enzymatic Dextran High MW Dextran Medium MW Dextran Indigenous Cane Polysacch. Oligosaccharides e.g., kestoses Bagacillos High MW Dextran Medium MW Dextran Low MW Dextran Long and Complicated Expensive and Imprecise Not Specific and Difficult to Interpret (False Positives) Sucrose Haze Dextran

From: Eggleston and Huet (2012). Zuckerindustrie.  Cane juice follows same pattern

 ~ 60 U.S. cents per analysis Cost of Mannitol Enzymatic Method  (~45 US cents NAD,12.47 US cents MDH)  Megazyme TM kit is US $4 per analysis  Biosentec TM kit (France) is US $4.65 per analysis

Alternative is To Use HPLC at the Factory Sample Mannitol Glucose FructoseMannitol Glucose Fructose RS Ratio ID Brixppm %/Brix F/G Crusher 7 am Crusher 11 am 18.30n.d Crusher 1 pm Mixed J Clear Dorr Clear Grav Clear Grav Syrup A Syrup B Final Mol 5 am Final Mol 9 pm Alma Plantation LLC. Lab Report Date: 8 Nov 2015 n.d.=not detected >0.2% to 0.33%/Brix of mannitol in juice means problems in factory and

HPLC at the Factory Sample Mannitol Glucose FructoseMannitol Glucose Fructose RS Ratio ID Brixppm %/Brix F/G Crusher 7 am Crusher 11 am 18.02n.d Mixed J Clear Dorr n.d Clear Grav n.d Clear Grav n.d Syrup B Syrup C Final Mol 5 am Final Mol 9 pm Mass Mass 91.62n.d Alma Plantation LLC. Lab Report Date: 7 Nov 2015 n.d.=not detected

Conc. Dextranase 4.6X dil Non-Conc. dextranase Dextran with no dextranase added isomaltotriose isomaltose Future Additions 40 ̊ C From: Eggleston et al. (2005). Process. Biochem.  Can be used to monitor the effect of enzymes at the factory

Future Additions  Can be used to monitor the effect of enzymes at the factory Min uC S mannitol leucrose isomaltose 1-kestose isomaltotriose ethanol Days Post-freeze GF  Can be used to monitor the deterioration of cane outside the factory and juice inside the factory From: Eggleston, Legendre and Tew (2004). Food Chem.

“ We were really blind to what was going on in the factory” “We were shooting everywhere without any direction” Belisario Montes, Alma factory, 12/8/2015 “You only waste time and dollars if you don’t take care of basics first” David Stewart, Alma factory, 12/2015