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

2. 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.

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

4. 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

5. 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

6. 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

7. 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.

8. 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

9. 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

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

11. 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.

12. 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

13. 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

14. 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

15. Comparing with other techniques
IR and FTIR Differential-pH
No sample treatment = no sample treatment
Time/test: 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 For analysis of fat, protein and lactose in milk

16. 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

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

18. 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 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.

19. 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.

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

21. 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.

22. 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.

23. 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