WHAT YOU NEED

• Visible Spectrophotometer

• Micropipettes▫ 10 - 100µL▫ 100 - 1000µL

• Cuvettes

• Parafilm

• Timer

WHAT TESTS CAN BE DONE

• Acetic Acid

• Amino Acid Nitrogen

• Ammonia

• Citric Acid

• Gluconic Acid

• Glucose & Fructose

• Lactic Acid

• Malic Acid

• Sucrose

WHAT TESTS CAN BE DONE

• Acetaldehyde

• Ascorbic Acid

• Ethanol

• Formic Acid

• Glycerol

• Metals

• Mannitol

• Sorbitol

• Succinic Acid

• Sulphite

• Starch

• Tartaric Acid

• Many more…

HOW THE KITS WORK

• Colorimetric

▫ Amino Acid Nitrogen (NOPA)

• Enzymatic

▫ Reduction of NAD+ to NADH

Malic Acid, Glucose & Fructose, Acetic Acid

▫ Oxidation of NADH to NAD+

Ammonia, Citric Acid

HOW THE KITS WORK

GOOD LABORATORY PRACTICE

• Calibrate Spectrophotometer

• Calibrate Micropipettes

• Cuvettes

• Kit storage

• Sample preparation

• Correct technique

SPECTROPHOTOMETER

• Calibrate every 3 months according to AS 3753

▫ Photometric accuracy

▫ Linearity check

▫ Stray light check

▫ Cell match check

SPECTROPHOTOMETER

• Erratic readings or excessive drift

▫ Dust in the sample compartment

▫ Fluctuating power supply

▫ Filter problem Go to 550nm, select %T and zero instrument on air

Adjust to 340nm and record %T, <5% is a problem

▫ Lamp re-adjustment required Go to 550nm and place white paper in light path. A bright

green rectangular light spot should be observed.

SPECTROPHOTOMETER

MICROPIPETTES

• Calibrate every 3 months according to AS 2162.2

▫ Distilled water and 4-figure balance required

▫ Test micropipette at its maximum capacity and again at close to its minimal volume (no more than 20% of its total capacity)

MICROPIPETTES

• Before calibrating, clean and regrease the micropipette piston

• Avoid liquid leaking inside the micropipette

▫ Always hold the micropipette more than horizontal

▫ Store on a micropipette rack rather than laying on bench or in a drawer

CUVETTES

• Disposable cuvettes should be disposed

• Do not handle the optical surfaces

• Avoid wiping the optical surfaces with tissue

• Do not clean quartz or glass cuvettes with detergent

KIT STORAGE

• Store kits cold to avoid reduction of shelf life

• Bring all reagents and samples to be analysed to room temperature prior to analysis

• Follow kit manufacturers guidelines for individual reagent storage…

KIT STORAGE

32 Brasser Avenue Dromana Victoria 3936 Australia T +61 3 5987 2242 F +61 3 5987 3303 E info@vintessential.com.au W www.vintessential.com.au

ABN: 60 068 057 045

ENZYMATIC ANALYSIS KIT FOR THE DETERMINATION OF L-MALIC ACID IN GRAPE JUICE AND WINE

PRODUCT Product no. 4A160, for 30 tests, for in vitro use only. PRINCIPLE OF MEASUREMENT L-malic acid is found in grape juice and wine and is determined enzymatically according to the following equations: MDH L-malate + NAD

+ ↔ Oxaloacetate + NADH + H

+

L-malic acid is oxidised by nicotinamide adenine dinucleotide (NAD) to oxaloacetate using L-malate dehydrogenase (MDH) enzyme as a catalyst. The equilibrium does not favour formation of oxaloacetate and so oxaloacetate is removed by a trapping enzyme. The amount of NADH formed is measured at 340 nm and is stoichiometrically related to the amount of L-malate consumed. In this method, glutamate oxaloacetate transaminase (GOT) is used as the trapping enzyme. In the presence of L-glutamate, the oxaloacetate is irreversibly converted to L-aspartate.

GOT Oxaloacetate + L-glutamate → L-aspartate + α-ketoglutarate CONTENTS The kit includes the following reagents:

Reagent No. Reagent Preparation Quantity Stability

1 Buffer Nil 33 mL 2 years at 4oC

2 NAD Add 6.6 mL of distilled water, mix to dissolve

6.6 mL 2 years at 4oC

(diluted: 1 year at 4oC,

2 years at -20 oC)

3 GOT Mix gently by inversion before use 0.4 mL 2 years at 4oC

4 MDH Nil 0.4 mL 2 years at 4oC

5 Standard Nil 3.3 mL 2 years at 4oC

The shelf life of Reagents 1 & 2 can be extended by placing aliquots in a freezer. Do not freeze enzyme reagents 3 & 4. Failure to store reagents at the recommended temperature will reduce their shelf life. For concentration of Standard, refer to label on bottle. SAFETY

Wear safety glasses Reagent 1 is mildly corrosive Do not ingest Buffer or Standard as they contain sodium azide as a stabilizer

SAMPLE PREPARATION• Bring sample to room temperature prior to analysis

• Decolourise highly coloured samples▫ PVPP▫ Activated carbon not recommended for some assays▫ Complete decolourisation not necessary

• Dilute samples to approximate concentration of kit standard

• Filter turbid samples

• Avoid excessive use of SO2

CORRECT TECHNIQUE

• Analyse a blank and kit standard in every run

• Zero spectrophotometer according to kit instructions, usually against air without cuvette in light path

• Be consistent in measuring reaction times between blank, standard and samples

• Spike & duplicate analysis

CORRECT TECHNIQUE

• Disposable micropipette tips are single use only

• When mixing cuvette contents invert gently, don’t shake. Same goes for reagent bottles

• Avoid cross-contamination between assays and especially contamination of reagents

• Purity of distilled water is important, decant fresh each run and discard after use

TROUBLESHOOTING

• Low recovery on kit standard

▫ Blank assay activity is a sign of contamination and will cause low standard and sample results

▫ Keep a logbook of absorbance values for the blank and standard assays for each kit

▫ Pipetting error

▫ Coenzymes may have perished

TROUBLESHOOTING

• High recovery on kit standard

▫ Check photometric accuracy of spectrophotometer or absorbance drift

▫ Volumetric accuracy of pipettes

▫ Check that the change in absorbance in the Blank assay is consistent with previous runs

▫ Human error (pipetted twice into the same cuvette?)

TROUBLESHOOTING

• Negative results

▫ If the sample contains negligible amounts of the compound being measured and a dilution factor has been applied, then negative values may be exaggerated.

▫ Excessive potassium metabisulphite interferes with malic acid assay (false negative results)

TROUBLESHOOTING

• Assay absorbance

▫ The higher the absorbance value, the lower the amount of light transmitted and measured by the photometer

▫ An absorbance of 1.000 occurs when 90% of the light has been absorbed

▫ At absorbance readings greater than 1.000 – 1.500 errors can occur because absorbance values increase exponentially

TROUBLESHOOTING

TROUBLESHOOTING

Or, simply call the manufacturer!

Details for Greg Howell

E: greg@vintessential.com.au

W: www.vintessential.com.au