Questions 72 - 79

1. Questions 72 - 79 Colorimetric

2. 72. Finding concentration of top standard point • What is the amount of tyrosine in Tube #6? (hint: Stock solution of Tyrosine 1mM=1nmol/μl, added 500 μl into tube #6) • 500 nmol • 0.05 mmol • 0.029 mmol • 50 nmol • 0.29 mmol Correct, 1nmol/ul x 500μl tube#6 =500nmol Incorrect, out by a factor 100000, mmol → nmol Incorrect, random number Incorrect, out by a fact of 10 Incorrect, random number

3. Correct, gradient of the line of equation; y=0.1989x-0.196 Not correct!!! A trap!!! Listen to the narration for an explanation 73. Working out extinction coefficient • What is the approximate E450nm (mM-1 cm-1) for the coloured compound formed in the assay; (Look at equation of the line on standard curve) • 0.199 • 250 • 4 • 2 • 0.196 40 9 107 38 12 Incorrect, random number Incorrect, random number Incorrect, random number Incorrect, y-intercept This is correct… divide the absorbance of any of the samples in the standard curve by their concentration…

4. 74. Minimum concentration measured by standard curve • What is the minimum concentration of tyrosine that you could reliably measure in an unknown solution? [hint: tube #2 added 100ul=100µM=Abs 0.21, want Abs 0.1) • ~0.02 µM • ~1 µM. • ~5 µM • ~50 µM • ~200 µM Incorrect, ↓concentration= Abs < 0.1 unreliable 22 20 21 109 34 Incorrect, ↓concentration=Abs <0.1 unreliable Incorrect, ↓concentration=Abs <0.1 unreliable Correct, Abs 0.21=100μM, Abs ~0.1= 50 μM Incorrect, abs ~0.4 Incorrect, abs ~0.4 See notes section (in powerpoint, beneath the slide) for an alternative explanation

5. 75. Using Optical fibers to measure Abs of standard curve • Which statement is CORRECT? • Using the new machine, the absorbance of the contents of Tube 6 will be greatly increased • Using the new machine, the extinction coefficient of the coloured compound will be decreased • This new machine will be able to give absorbance readings to at least FIVE decimal places with great accuracy • The new machine will be able to determine if tyrosine itself absorbs at 460 nm • The new machine will enable us to create a workable standard curve with 100‑fold smaller volumes 11 11 33 15 144 Incorrect, amount of tyrosine has not changed Incorrect, amount of tyrosine has not changed Incorrect, information not given Incorrect, tyrosine doesn’t absorb at 460nm Correct, original assay 2ml, using optical fiber 20μl

6. 76. Lowed Abs of standard curve • Your assistant repeats your set up but, the slope of their standard curve is 80% of yours. In your diagnosis of their mistake, what can you RULE OUT (ie, what would NOT have caused this)? • They measured the samples at the wrong wavelength • They used a pipette for the tyrosine solution that consistently delivered 20% less than it was meant to • They used a pipette for the tyrosine solution that consistently delivered 100 µl too little • They incubated their tubes for too short a time • They incubated their tubes for too long 22 21 33 22 111 Yes, if read not λmax ↓ Abs Yes, ↓ tyrosine 20% ↓ Abs Yes, ↓ tyrosine ↓ Abs TRAP… listen to the narration!!! Yes, not enough time for the reaction to go to completion, ↓ Abs Rule out

7. 77. Unknown tyrosine concentration • One of your colleagues has made up a stock tyrosine solution but cannot remember its concentration (Refer to Table); Which statement is CORRECT? • This data shows that the real relationship between absorbance and concentration is complex and cannot be solved with a simple linear regression • The concentration of your colleague’s solution is too high for you to determine using your assay • Your colleague’s solution is too dilute for you to determine using your assay • Your colleague’s solution contains something that interferes with your assay • These results enable you to give your colleague a reasonable estimate of the concentration of their solution 9 109 8 18 44 Incorrect, Abs >1 Correct, with readings obtained can’t determine concentration accurately Abs >1 Incorrect, 4 out of 5 Abs >1, not reliable Incorrect, can’t be certain Incorrect, 4 out of 5 Abs >1, not reliable

8. 78. Using real plasma • How could you control for the extra problems associated with using plasma? • The complete absorption spectrum of each plasma sample needs to be determined before the assay can be done • There is no need to introduce any extra controls because the pink colour or milkyness will not affect the absorbance at 450 nm • If samples look pink or milky we should just use smaller volumes of these plasma samples in the assay • Blanks containing plasma but no Solution A or B need to be set up • All the standards need to be spiked with plasma samples before assay 55 10 17 85 44 Silly, waste of time Pink colour or cloudiness will affect Abs reading Abs need to lie between 0.1-1, ↓volume ↓ amount of tyrosine ↓abs Correct, if volume is replaced by water Standard curve needs to have known concentration of tyrosine

9. 79. Concentration of unknown • Their samples of plasma are precious and they do not want you to use any more than you need. What do you tell them? (hint: normal 80µM, fatigue syndrome ~60% higher→128µM. Standard curve 50-250µM) • “Easy. I only need to use a total of 10 µl of each sample to get accurate enough measurements to see the difference” • “My current assay is sensitive enough to get accurate results with just 10 µl of each sample, but the differences you’re expecting to see are just too small” • “I’ll need to use 0.5 to 1 ml of plasma in each assay tube, so by the time I’ve done replicates and controls, I may have used several mls of your plasma” • “It’s impossible. Even if you buy me one of those new fibre-optic machines” • “The changes that you anticipate cannot be detected by any colorimetric method” 65 51 68 7 11 Samples are not concentrated effort, 10µl won’t obtain Abs 0.1-1 Assay is sensitive enough BUT not with 10µl Correct, volumes between 0.5-1ml will give Abs 0.1-1 Possible, optical fibers will reduce volume needed of plasma Silly, changes between patients can be differentiated