WHAT YOU NEED. Visible Spectrophotometer. Micropipettes. Cuvettes. Parafilm. Timer µL

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2 WHAT YOU NEED Visible Spectrophotometer Micropipettes µL µL Cuvettes Parafilm Timer

3 WHAT TESTS CAN BE DONE Acetic Acid Amino Acid Nitrogen Ammonia Citric Acid Glucose & Fructose Lactic Acid Malic Acid Sucrose Gluconic Acid

4 WHAT TESTS CAN BE DONE Acetaldehyde Ascorbic Acid Ethanol Formic Acid Glycerol Metals Mannitol Sorbitol Succinic Acid Sulphite Starch Tartaric Acid Many more

5 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

6 HOW THE KITS WORK

7 GOOD LABORATORY PRACTICE Calibrate Spectrophotometer Calibrate Micropipettes Cuvettes Kit storage Sample preparation Correct technique

8 SPECTROPHOTOMETER Calibrate every 3 months according to AS 3753 Photometric accuracy Linearity check Stray light check Cell match check

9 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.

10 SPECTROPHOTOMETER

11 MICROPIPETTES Calibrate every 3 months according to AS 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)

12 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

13 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

14 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

15 KIT STORAGE 32 Brasser Avenue Dromana Victoria 3936 Australia T F E info@vintessential.com.au W ENZYMATIC ANALYSIS KIT FOR THE DETERMINATION OF L-MALIC ACID IN GRAPE JUICE AND WINE ABN: 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 4 o C 2 NAD Add 6.6 ml of distilled water, mix to dissolve 6.6 ml 2 years at 4 o C (diluted: 1 year at 4 o C, 2 years at -20 o C) 3 GOT Mix gently by inversion before use 0.4 ml 2 years at 4 o C 4 MDH Nil 0.4 ml 2 years at 4 o C 5 Standard Nil 3.3 ml 2 years at 4 o C 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

16 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 SO 2

17 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

18 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

19 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

20 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?)

21 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)

22 TROUBLESHOOTING Assay absorbance The higher the absorbance value, the lower the amount of light transmitted and measured by the photometer An absorbance of occurs when 90% of the light has been absorbed At absorbance readings greater than errors can occur because absorbance values increase exponentially

23 TROUBLESHOOTING

24 TROUBLESHOOTING Or, simply call the manufacturer! Details for Greg Howell E: W: