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Supplier Showcase Seminar: QC & Enzymatic Analyses
Thursday, June 191:30 pm - 3:00 pm
Chair: Patricia Howe, Patricia Howe Wines, California Theme: Improve and understand your Malic and Glu/Fru
analysis Speakers:
Jason Gerard, Tom Eddy Wines, CaliforniaPatricia Howe, Patricia Howe Wines, CaliforniaSamantha Kollar, Vinquiry, CaliforniaMichael Moyer, Walla Walla Community College, Washington
Geoffrey Anderson, Unitech Scientific, California
ASEV Supplier’s Showcase, Portland OR
June 19, 2008
Improve and understand your Malic & Glu/Fru analyses
Issues in Automated Analyses
Geoffrey Anderson Geoffrey Anderson Unitech Scientific LLC Unitech Scientific LLC
Lakewood, CA Lakewood, CA
Visit us: Visit us: www.unitechscientific.com
Advantages of Automation
Efficiency: more data . . . Faster
Better Accuracy & ReliabilityMulti-point Standard Curves (improved
accuracy over analytical range)Verified Calibrations (Check Wines as Verified Calibrations (Check Wines as
controls, Levy Jennings Charts)controls, Levy Jennings Charts)
Systematic (fewer random errors)
MoreMore Advantages of Automation
EasierRandom Access - Random Access - multiple tests run
togetherEasy assay set-up Easy assay set-up Easy accurateEasy accurate reportingreportingCurve Editing Curve Editing Store Calibration Curves Store Calibration Curves
Managing the Issues
Troubleshooting – Common Themes
Reagent IssuesSpectrophotometers vs. AnalyzersDiscrete & Other Analyzers
Analyzer Components & Functions
Operating software 2 syringe pumps wash headwash headOptical Optical
systemsystem
samples reagents ReactionsDI water
Controlling Potential Sources of Error
Reagents, Standards Fluid Handling, Pipetting Photometric Analysis & Samples & cleanliness
ISSUE: Poor ‘splits’
Case Study #1(a) “many samples affected, results were good in the morning run”
RESOLUTION: check for air bubbles check for reagent spills (photometry issues)
ISSUE: Poor ‘splits’(extreme samples)
Case Study #1(b) “only ‘dry’ wines (or only juice samples) affected”
RESOLUTION: check ‘delta ABS’ (raw data) <0.030 or greater (> high Std ABS) consider alternate protocols
2 Protocols: Accuracy over a Broad Analytical Range
Sample Volumes
1) L-Malic ‘dry’ (for high sensitivity,
0.02 – 0.5 G/L) 2uL Sample volume
2) L-Malic ‘juice’ (detecting high levels,
0.2 – 5.0 G/L) 2uL Sample Volume
pre-diluted 10-fold
Problem Solving Approach: Isolate instrument issues
Instrument Diagnostics (example)
a) ‘Self Test’ Measure Precision & Accuracy (Pipette delivery of dye + diluent to cuvettes)
b) Blank (measure d.i. water in cuvette)
c) Photometer Voltage(s) (i.e. response to light at each wavelength)
Distinguish instrument vs. chemistry issues
Self Test (example)
Sunday, June 08, 2008, 11:01
Lamps - OKBlank - OK Small Sample Medium Sample Large
Sample Pipetting Accuracy: OK OK OK Photometer/Plate Positioning: OK OK OK PASSED (Blank out of recommended range)DETAILS:(6.3.1.162/1720/0.95/0.92)Temperature at start: 29.3 °C Temperature at end: 24.8 °CBlank Abs: 0.0016
Distinguish instrument vs. chemistry issues
Self Test (precision detail)
Type Target Mean SD %CV %Diff ------------------------------------------------------------------Large 2.4190 2.4918 0.0147 0.5898 3.0091Medium 1.3440 1.4202 0.0082 0.5756 5.6687Small 0.2690 0.3071 0.0034 1.0930 14.1654
ISSUE: Standard Curve
Case Study #2(a)
“LMA curve is OK, G/F curve has poor duplicates & poor r2 value”
RESOLUTION:Verify delta A values (replace G/F reagents)Consider editing G/F Standard CurveRun Self Test
Editing a Standard Curve5-Point Calibration Curve
r2 = 0.95972
Editing: Select Calibration Points
Editing a Standard Curve
4-Point Calibration Curve
r2 = 0.99997
ISSUE: Standard Curve
Case Study #2(b)
“Curve ‘falls-off’ at top end”
Likely Reagent Cause: Substrate depletion (Working Reagent) or weak enzyme activity
RESOLUTION: Replace G/F Reagents
Potential ISSUE: Reagent Stability (Reagents may be left ‘on-board’)
RESOLUTION: Anticipate Deterioration & Dilution
- Schedule - Fresh Working Reagents - Use Enzyme ALIQUOTS & Standards ALIQUOTS - Control Temperature as required
ISSUE: Standard Curve
Case Study #2(c) “Everything was great Friday, nothing looks good Monday”
Verify change in system
RESOLUTION:Weekly P.M. (e.g. Self Test, check tubing, etc.)
Check for air bubbles, clean supply water, clean cuvettes, etc.
ISSUE: Check-wine is ‘Out of Range’
Case Study #3 “D-Fru is low, D-Glu is OK”
Note: D-Fru reaction may not be complete
(PGI Reaction is the rate-limiting step)
RESOLUTION:
Load fresh PGI aliquot
ISSUE: ‘Self Test’: Filter Voltage fails
RESOLUTION: Photometer P.M.Photometer P.M.Check & clean lamp(s)Check & clean lamp(s)Clean Photometer(s) lense(s)Clean Photometer(s) lense(s)Clean any reagent spillsClean any reagent spillsCheck cuvettes for cleanliness & Check cuvettes for cleanliness &
scratchesscratches
Optical DesignOptical Design
4 optical channels
align with 4 microwells
and read each well at
all 8 wavelengths
(340-750nm)
Quality ControlQuality ControlTrackingTracking
LIS compatibleLIS compatible
Preventive Maintenance for Accurate Automated Results
A. Fluid Handling System A. Fluid Handling System (e.g. bubbles)(e.g. bubbles)
B. Photometry System B. Photometry System (lamp voltage, (lamp voltage, spills etc.)spills etc.)
C. System Diagnostics C. System Diagnostics (self test)(self test)
D.D. Daily/WeeklyDaily/Weekly (prime, bleach or change (prime, bleach or change tubing, water supply, confirm performance)tubing, water supply, confirm performance)
Data Issues
Sample Data base (avoid transcription errors)
Review results on-screen (Accept or Rerun)
Sorting Data, Print & Distribute Reports & export reports (avoid transcription errors)
Issues in Automated Analyses
Geoffrey Anderson Geoffrey Anderson Unitech Scientific LLC Unitech Scientific LLC
Lakewood, CA Lakewood, CA
Visit us: Visit us: www.unitechscientific.com