Question

How do I make a line of best fit (regression) with the following data? I am aware of how to make it in Microsoft Excel, but I am having trouble figuring out how to find my x value for making the graph. Y value is absorbance, what do I use for x value to calculate the slope?

Tube 0.5 mM sulfide standard (ml) Sulfide amount (umol) Sulfide reagent (ml) 0.0 4.9 0.11 0.00 0.2 FeCl3 dH2O to Absorbance 10ml total (ml) (670nm) (ml) 5 4.8 0372 4.6 / 10.502 0. 11 4.4 0.650 0.1 - 4.2 0.734 0.1 0.17 0.4 0.6 0.8 Make sure that the spectrophotometer has warmed up for at least 10 minutes and set the wavelength at 670nm. Pour a small sample from tube 1 into the cuvette and place the cuvette into the spec. Set the absorbance to 0.00 to blank the machine. 4 Measure the absorbance of the other standards, rinsing the cuvette with distilled water between each sample. The absorbance for all the tubes should read less than 1.2 absorbance units (AU). 5. To measure the absorbance of your Winogradsky sample, add 0.1 ml of the sample to a sulfide reagent tube. Add 4.9ml of dH20 and 0.1 ml of FeCl. Mix well and wait at least 3 minutes for color development. 6. If the blue color falls within the color range that you observed for the standard curve samples: wait another 7 minutes before measuring the absorbance. If color is darker than any of the standard curve samples: prepare another reagent tube with less sample added (0.01 to 0.05ml) using a P20 Pipetman. Add 4.9ml of dH20 and 0.1 ml of Fech. Mix well and let color develop for 10 minutes before measuring the absorbance. If color is lighter than any of the standard curve samples: make another reagent tube with more sample (1 to 5ml). Add 0.1ml of FeCls and make up to 10ml with dH20. Mix well and let color develop for 10 minutes before measuring the absorbance. Do NOT discard your first sample just because you're making another one. Wait the full 10 minutes and measure it. If the absorbance falls within the standard curve, it's a good measurement and you can use it to determine sulfide concentration. 7. Refer to the sample calculations on the next page for instructions on how to convert absorbance readings into mM sulfide. Collect data from other members of your bench and fill in the table on page 82.

Answer 1

Given the concentration of sulfide is 0.5 mM. i.e. 1 liter of sulfide contains 0.5 milli moles (which is 500 micromoles).

Thus 0.2 ml of the sulfide sample will contain = 0.2 * 500/1000 = 0.1 micromoles

Similar calculation is done for other amounts of sulfide

Tube	0.5mM sulfide standard (ml)	Sulfide amount (micromoles)	Sulfide (reagent (ml)	FeCl3 (ml)	H2O (ml)	Total volume (ml)	absorbance
1	0	0	4.9	0.1	5	10	0
2	0.2	0.1	4.9	0.1	4.8	10	0.372
3	0.4	0.2	4.9	0.1	4.6	10	0.502
4	0.6	0.3	4.9	0.1	4.4	10	0.65
5	0.8	0.4	4.9	0.1	4.2	10	0.734

Plotting Absorbance as a function of sulfide amount (miromoles), we get,

Absorbance = 2.087* sulfide amount R? = 0.865 Absorbance Series1 - Linear (Series1) 0 0.05 0.1 0.35 0.4 0.45 0.15 0.2 0.25 0 .3 Sulfide amount (micromoles)