Transcript Enzymatic analyses on wine

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
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Sample numbers
Sample volume available for analysis
Time available
Operator skills
Funds available
Instrumentation available
Micropipettes
• Hand method: Very important
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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:
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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 board @ 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
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Time consuming : Maximum 12-14 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
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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 ±R800 000
• 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
Thank you