EC Line and the CL-10 Plus System Product Training

The EC- Line Product Range • CL-10 PLUS Instrument using the proprietary differential-pH technology. • EC- Line Reagents. EC-Line is the range of reagents and diagnostics to be used with CL-10 instrument.

The differential pH principle Let’s study the differential pH principle using the Urea Test in Milk.

The components of the CL-10 Plus System Instrument: Two capillary glass electrodes to measure pH Mixing chamber Peristaltic pumps Tubings that faciliate transfer of reagents and samples in the instrument Kits and supplies (EC-Line): (Urea Milk) Polif solution (Wash Solution) Regenerating Solution Strong regenerating solution (Urea Kit) Reagents for the enzymatic reaction. Shelf life: 12 months

Basic components of the instrument Left: Where solutions/samples get loaded into the instrument Right: Where solutions/samples exit and enter the waste bottle Waste Bottle Resting: Polif Solution Testing: R1 Diluent Mixing chamber Where milk samples are pipetted into Resting: Distilled Water Testing: Urease Enzyme

Pipetting into the reaction chamber Technique for pipetting into the reaction chamber. Load sample into pipette Insert pipet into reaction chamber Wait until you can feel the stirbar stirring Depress pipette all the way to release sample and keep it depressed as you lift the tip out of the chamber *This prevents fluid from being drawn out of the reaction chamber, affecting the reaction *Pipette tips are re-usable, wipe the outside of the pipette gently before inserting new samples

The Urea Reaction Urease is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The Reaction Urea + 3 H2O  2NH4+ + CO2 + 2 OH- H2O + CO2  HCO3- + H+ Urease As the reaction proceeds, the level of H+ will increase, the pH will decrease. As indicated in the reaction, the number of H+ ion is directly proportional to concentration of Urea.

dpH=0 Injecting Sample pH Buffer + Sample D2-D1 Ac CoA Electrodes Micropipette Containing Sample Time (s) D1 D2 pH D2-D1 dpH=0 Ac CoA D2 Vial containing Enzyme (Urease) Electrodes Buffer + Sample Mixing Chamber Stir Bar D1

Enzyme-Urease being pumped to one of the electrodes (D2) Tempo (s) D1 D2 pH D2-D1 dpH=a Ac CoA D2 Mixing Chamber Enzyme- Shown in Yellow being pumped into D2 D1

Starter (enzyme /substrate) The Reaction Kinetics mpH Buffer + sample Starter (enzyme /substrate) D1 Enzymatic Reaction D2 Time (s)

What are D1, D2 and mpH? D1 = Difference in mpH measured by 2 electrodes filled with the same solution (buffer + sample) D2 = Difference in mpH measured by 2 electrodes when Electrode 1 has the same solution of D1 and Electrode 2 has D1 solution + enzyme mpH = Difference between D2 and D1 mpH is driven by the enzymatic reaction.

Why this technique? The technique is based on an enzyme reaction The measurement is not affected by interference from ammonium Compared to traditional UV methods there is no color, opalescence and micro-particles interference High-tech micro-flow electrodes allow accurate, reproducible measurements These produce a repeatable, reliable, and accurate test method that has been adopted by many laboratories worldwide Eliminate results subjectivity because there is no need to “interpret results” Test results are recorded and can be exported to Excel or printed out directly from the computer hooked up to the instrument

Comparing with other techniques Traditional techniques: Titrators, refractometers, pH-meters Differential-pH Sample treatment = No sample treatment Colour interference = No colour interference Preservatives interference = No preservatives interference Time consuming = Faster Operator dependency = Operator independent Limited application panel = Wider application range Aspecific = Highly specific Traditional techniques have lower specificity and lower accuracy Refractometers- Cannot differentiate between different sugars Automated pH meters and titrators Manual Titration

Comparing with other techniques Spectrophotometers (UV) Differential pH Sample treatment needed = No sample treatment Rather fast = Equally fast or slightly slower Short linearity range = Wider linearity Colour interference (auto-treatment) = No interference Possible Matrix effects = No effect for tested matrices Some parameters not reliable = Reliabile for tested matrices An autospectrometer

Comparing with other techniques IR and FTIR Differential-pH No sample treatment = no sample treatment Time/test: 120-400 test/hour = 15 sec/test to 5 minutes Expensive Instrument = Medium to lower cost instruments Expensive maintenance = Less expensive to maintain WineScan from FOSS MilkoScan from FOSS Bentley 2000- For analysis of fat, protein and lactose in milk

Comparing with other techniques HPLC Differential-pH Requires sample treatment = No sample treatment Very accurate and precise = Equal and comparable Time/test 5 to 20 minutes = 15 sec/test to 5 minutes Expensive columns = Same equipment for all tests types Expensive maintenance Dangerous waste = Disposable waste Universal use, versatile = Limited application/matrices panels

Quality Indicators and Applications in Milk Urea Lactose L-Lactic Acid Titratable Acidity Alkaline Phosphatase

Quality Indicators and Applications in Milk UREA (ISO Std 14637) In most countries urea is included in the milk payment scheme. Maximize feed levels High urea concentrations  high protein level Low urea concentrations  low protein level Urea analysis in milk is important because it is related to the animal’s health. Levels of urea in milk stay in the range 24-33 mg/100 cc. LACTOSE Lactose occurrs naturally in high concentrations in milk (≈130 mM). Also in the payment scheme, in some countries. In low-lactose milk products, this is also analysed for consumers with possible allergy/intolerance.

Quality Indicators and Applications in Milk L-LACTIC ACID Indicator of milk freshness. Fresh milk contains little to no Lactic acid. Milk for high quality production must contain maximum 30 ppm Lactic acid. Process control during fermentation in the yoghurt industries. In fermented milk and cheese products, the determination of D and L isomer forms of lactic acid is very important. Ratio affects taste and aroma. TITRATABLE ACIDITY Indicator for milk freshness. ALKALINE PHOSPHATASE Alkaline phosphatase enzyme is naturally found in milk. Its content in raw milk depends on breed, season and lactation phase of the animal. The enzyme is inactivated at the same temperature conditions where pathogens micro-organisms are destroyed. Its absence in milk is an indication of a successful pasteurization.

Quality Indicators and Applications in Wine, Must and Cider pH Sucrose Glucose Fructose Total acidity Glycerol Citric Acid Acetic Acid L-Lactic acid L-Malic Acid

Quality Indicators and Applications in Wine GLUCOSE/FRUCTOSE Control of sugars in grapes and wine or must gives the alcoholic potential in final product. TOTAL ACIDITY Allows correction if de-acidification is needed. Allows monitoring of malic-lactic fermentation. Improves the taste of wine. pH also affects the color of wine. L-MALIC Malic Acid is naturally present in must and develops into L-Lactic Acid in wine by an enzymatic reaction (Malolactic Fermentation). If not controlled, it could also turn into Acetic Acid, which is very dangerous!). L-LACTIC Monitoring a balance between malic and lactic acid controls taste. Affects properties of the final product.

Quality Indicators and Applications in Wine ACETIC ACID (the most abundant of volatile acids) An unwanted and dangerous compound. Corrections are possible at initial stages only! Above 1.08 g/l for white wines and 1.20 g/l for red wines, the product is not allowed to be sold as table wine. SUCROSE Sucrose does not naturally exist in wine, thus can be added for special fermentations (Champagne production) or as an Ethanol developer.

CL-10 Instrument and EC Line Reagents Summary Comprehensive system and reagents for process and quality control Requires no pre-sample treatment Ready-to-use reagents Fast and reliable No sample centrifugation Rapid Assay Official ISO-IDF method