To determine an unknown concentration of glucose; Glucose-6-phosphate deyhdrogen

Question

To determine an unknown concentration of glucose; Glucose-6-phosphate deyhdrogenase converted glucose-6-phosphate (G6P) to 6-phosphogluconate. Durring the process, one molecule of NAD+ will be reduced to NADH. While 6-phosphogluconate cannot be measured using a spectrophotometer, the amount of NADH can be measured at 340 nm. This will allow you to determine the amount of glucose in the original sample.

Glucose sample was diluted to 1:20 ( 2 microliters of Glucose and 38 microliters of water for a total volume of 40 microliters of sample).

Tube contains 1000 microliters of glucose reagent and 20 microliters of the total 40 microilters of glucose dilution sample. (Total volume = 1020 microliters or 1.02 mL). Absorbance is .132.

Concentration of NADH was found by using beer's law using 6.22 mM cm-1 as extinction coefficient. Concentration of NADH = .0212 mM.

Moles of NADH that are present in 1.02 mL sample was found by : .0212 mM x 1.02 mL = .021624 mM/mL

How do i find th e concentration of glucose in the original unknown glucose sample? Note: remmber the original unknown sample solution was diluted)

Explanation / Answer

Given in the tube,

Glucose = 0.001 ml

Water = 0.019 ml

NADH = 0.21624 mM/ml

So amount of NADH in 1.02 ml = 0.22 mM

So original conc of glucose = 0.221 ml

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