EC Line and the CL-10 Plus System Product Training.

EC Line and the CL-10 Plus System

Product Training

The EC- Line Product Range

• CL-10 PLUSInstrument using the proprietary differential-pH technology.

• • EC- Line 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.

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

The components of the CL-10 Plus System

Basic components of the instrument

Left: Where solutions/samples get loaded into the instrument

Resting: Distilled Water

Testing: Urease Enzyme

Right: Where solutions/samples exit and enter the waste bottle

Waste Bottle

Mixing chamber

Where milk samples are pipetted into

Resting: Polif Solution

Testing: R1 Diluent

Pipetting into the reaction chamber

Technique for pipetting into the reaction chamber.

1) Load sample into pipette

2) Insert pipet into reaction chamber

3) Wait until you can feel the stirbar stirring

4) 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.

Time (s)

D1

D2

pH

D2-D1

Ac

CoA

dpH=0

Mixing Chamber

Micropipette Containing Sample

Vial containing Enzyme (Urease)

D1

D2

Electrodes

Stir Bar

Injecting Sample

Buffer + Sample

Tempo (s)

D1

D2

pH

D2-D1

Ac

CoA

dpH=a

Enzyme- Shown in Yellow being pumped into D2

D2

D1

Enzyme-Urease being pumped to one of the electrodes (D2)

Mixing Chamber

Time (s)

The Reaction Kinetics

D1

D2

Buffer + sample Starter (enzyme /substrate)

Enzymatic Reaction

mpH

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

Traditional techniques:Titrators, refractometers, pH-meters Differential-pH

1. Sample treatment = No sample treatment2. Colour interference = No colour interference 3. Preservatives interference = No preservatives interference4. Time consuming = Faster5. Operator dependency = Operator independent6. Limited application panel = Wider application range7. Aspecific = Highly specific

Traditional techniques have lower specificity and lower accuracy

Comparing with other techniques

Manual Titration Automated pH meters and titrators

Refractometers- Cannot differentiate between different sugars

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

Comparing with other techniques

An autospectrometer

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

Comparing with other techniques

WineScan from FOSS MilkoScan from FOSS Bentley 2000- For analysis of fat, protein and lactose in milk

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

Comparing with other techniques

Quality Indicators and Applications in Milk

Urea

Lactose

L-Lactic Acid

Titratable Acidity

Alkaline Phosphatase

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 ACIDITYIndicator for milk freshness.

ALKALINE PHOSPHATASEIndicator for milk freshness.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 Milk

pH

Sucrose

Glucose

Fructose

Total acidity

Glycerol

Citric Acid

Acetic Acid

L-Lactic acid

L-Malic Acid

Quality Indicators and Applications in Wine, Must and Cider

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.

SUCROSESucrose does not naturally exist in wine, thus can be added for special fermentations (Champagne production) or as an Ethanol developer.

Quality Indicators and Applications in Wine

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