Date:

Trainee:

Need the following materials:

A	B
Microtube rack
17 X 2 mL microtubes
MilliQ water container
10 uL pipet
100 uL pipet
1000 uL pipet
10 uL pipet tip
100 uL pipet tip
1000 uL pipet tip
Vortex mixer
Sharpies
Primary stock solution of KH2PO4
MilliQ

How to aspire and dispense with pipet

(a) When aspiring solution, ensure the pipette to be held vertically.

(b) When dispensing, ensure you hold the pipette at an angle (10-45°). Working to these angles ensures the desired liquid amount is drawn into the tip properly and that all of the liquid is fully dispensed without leaving any residue in the tip.

Standard working solutions

A	B

A	B	C	D	E
S1	0		500
S2	2		498
S3	5		495
S4	10		490
S5	25		475
S6	50		450
S7	75		425
S8	100		400

Need the following materials:

A	B
50 mL Falcon tube (3)
Transfer pipet (1)
Scoopula (1)
Anti-static weighting plate (2)
MilliQ
Kimwipe
Chamber balance
Graduated cylinder (1X25 mL)
Goggles

2.5% ammonium molybdate reagent

In an anti-static weight plate, weigh ~0.25 g ammonium molybdate, transfer into the labelled falcon tube.

A

Measure 10 mL MilliQ (use transfer pipet for the final topping)

Rinse the residue on the weighing plate into the falcon tube with the MilliQ.

Transfer all remaining MilliQ into the falcon tube

10% ascorbic acid reagent (avoid light exposure)

In an anti-static weight plate, weigh ~1 g ascorbic acid, transfer into the labelled falcon tube.

A

Measure 9 mL MilliQ (use transfer pipet for the final topping)

Rinse the residue on the weighing plate into the falcon tube with the MilliQ.

Transfer all remaining MilliQ into the falcon tube

Sulfuric acid reagent:

Measure 17 mL MilliQ (use transfer pipet for the final topping) and transfer into a Falcon tube

Carefully add 1mL 18 concentrated sulfuric acid into the water

Vortex ammonium molybdate and ascorbic acid solutions.

Use graduated cylinder, measure and transfer 6 mL 2.5% ammonium molybdate reagent into the sulfuric acid tube.

Use graduated cylinder, measure and transfer 6 mL 10% ascorbic acid reagent into the sulfuric acid tube.

Vortex the reagent.

Preheat incubator/shaker to 37°C

Equipment

Value	Label
SHAKING INCUBATOR	NAME
71L	TYPE
Corning® LSE™	BRAND
6753	SKU

Add 500µL reagent to each standard working solution.

Vortex each tube.

Incubate at 37°C for 1h 0m 0s while shaking at 200 rpm

Load microplate with 250 uL reactant from each tube, duplicate.

Layout in the microplate:

A	B	C	D	E
A	S1	S1	S1	S1
B	S2	S2	S2	S2
C	S3	S3	S3	S3
D	S4	S4	S4	S4
E	S5	S5	S5	S5
F	S6	S6	S6	S6
G	S7	S7	S7	S7
H	S8	S8	S8	S8

Turn on microplate reader

Equipment

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU

Setup layout and method

A	B
Shake duration	00:00:05
Shaking type	Continuous
Shaking force	High
Shaking speed [rpm]	600
Wavelength [nm]	820
Measurement Time [ms]	100

Place microplate onto the plate holder

Read the plate

Export data to Excel sheet

In the Excel sheet, subtract the average absorbance at 820 nm of the blank standard replicates from the absorbance at 820 nm of all other standard working solutions.

This gives the corrected absorbance from each well.

Calculate the concentration of each standard working solution

Unit for each content:

Primary concentration g/L

Primary volume uL

MilliQ volume uL

Standard working solution g/L

Phosphate in standard working solution uM

Y-axis: Corrected absorbance

X-axis: Phosphate (uM)

Calculate slope ( S ), intercept ( b ), coefficient of determination (R²), standard deviation (S_y) of the absorbance from the four replicates of the blank, and LOD = 3.3 X S _y/ S

A	B
S
b
R2
Sy
LOD

What information does LOD provide?

Is it necessary to be aware of the order when mixing water with concentrated sulfuric acid?

Neutralize acidic chemicals with baking soda in a plastic beaker

Rinse microtubes and microplate with tap water

Dispose microtubes and microplate into recyclable garbage bin

Dispose pipet tips into sharp garbage container

Rinse graduated cylinder, Falcon tubes and MilliQ container with DI water, place them into a basket to air-dry