Enzymatic analyses on wine By Wernich Kühn Central Analytical Facility Stellenbosch University

Enzymatic methods Sugars (Glucose, Fructose, etc.) Organic acids (L-Malic acid, L-Lactic acid, Acetic acid, etc.) YAN (Yeast assimilable nitrogen) (Ammonia, Alpha amino nitrogen) Acetaldehyde Glycerol Etc…

Hand method vs automation The same enzymatic kit can be used for both automated and hand analyses Consider: Sample numbers Sample volume available for analysis Time available Operator skills Funds available Instrumentation available

Micropipettes Hand method: Very important Used during make-up of standard (if not commercially bought) Large volumes used makes commercial standards very expensive to use Used during analysis Analysts should be very efficient in their skill as this is crucial to the results Calibration records for pipettes should be kept up to date to ensure accuracy of results Consumables: large number of tips will be used to prevent contamination Advantage: training an analyst to use these is easy and affordable

Micropipettes Robot: Less important Used during make-up of standard (if not commercially bought) No micropipettes used during analysis Analyst pipetting skills less important – unless commercial standards are not used Less frequent calibration due to lesser use of pipettes Consumables: Very small number of tips used due to automation

Hand method using spectrophotometer

Contamination Hand method: Take care to prevent contamination Use new pipette tip for every step of analyses – no risk of contamination Risk of cross contamination of samples Risk of cross contamination of reagents - this may deactivate an entire bottle Risk of contamination of samples with reagents Risk of contamination of reagents with samples Risk of dilution of reagents with water when diluting samples Risk of contamination of water with samples after mixing with tip and consequent contamination of samples or reagents

Contamination Enzyme robot Lower risk Samples are poured into sample cups – no contamination risk Dispenser is washed after every dispense thus no risk of cross contamination of samples No risk for cross contamination of reagents No risk for contamination of samples with reagents No risk for contamination of reagents with samples No risk for dilution of reagents No risk for contamination of water

Stability of enzymes Kits are stable up to the last day of the month as indicated on the label Generally 6 months to 1 year – differs between kit types. Not ready to use reagents are less stable. Some have to be prepared fresh daily before analyses Different brand kits have different stability, find one to suit your needs If less stable: hand method advantageous. Large volumes means it is used prior to expiration date – great benefit Enzyme robot: often stable up to 30 days on 8°C

Hand method using spectrophotometer Advantages: Spectrophotometer, incubator and pipettes much less expensive than enzyme robot Smaller instrument takes less space than large robots Maintenance much less expensive on spectrophotometer compared to enzyme robot and can be performed by everyday analyst Training of analysts lower level of difficulty Larger volumes of reagent used means less risk of reagents passing expiry date and having to be disposed of

Hand method using spectrophotometer Disadvantages Time consuming : Maximum analyses per hour (operator dependant) Larger sample volumes are used Smaller number of analyses can be done per kit (48) Reagent cost per sample more expensive QC standard volumes used is larger – more expensive when using commercial standards (9ml standard – 90 analyses) Hand dilutions : Accuracy influenced by calibration of pipette (instrumentation accuracy) as well as human error (technique accuracy) Contamination risk if pipette tips are not replaced for each step of the method Calculations are done by operator, room for error

Automated method using enzyme robot Advantages Time saving: 100 analyses per hour Low volume cuvettes means small sample volumes for analyses Large number of analyses can be done per kit (400) Reagent cost cheaper than hand method Commercial standards less expensive than hand methods due to smaller volumes used (9ml standard – 1000 analyses) Automated dilutions – no human error Contamination risk minimized as robot washes needle after every injection Calculation software – no operator involved

Automated method using enzyme robot Disadvantages Enzyme robots quite expensive ±R Expensive maintenance by specialised technician Time aspect with regards to scheduled maintenance, technician not always available Time is also a problem with unexpected break-down. Analyst to be trained extensively – can take up to 1 year Consider number of analyses to be performed – small numbers of tests may mean than reagents expire before being used Method development difficult and time consuming Monthly calibration uses much of the reagents Cuvettes for analyses are very expensive

Enzyme robots

Kits

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