Phosphate Assay Analyzer

Determine the concentration and uncertainty of a phospholipid stock solution from a Bartlett inorganic-phosphate assay, following the Heberle Lab protocol SOP-PIASSAY-001 (full PDF). Enter the absorbance readings for your standard curve and your replicate samples below, then click Analyze. The tool fits a linear standard curve (floating intercept from your blanks), converts each sample's absorbance to phosphate, and reports the stock concentration with a 95% confidence interval that combines replicate scatter and standard-curve uncertainty. For most phospholipids (PC, PE, PS, PG, PI, PA, SM) there is 1 phosphate per lipid; cardiolipin has 2.

Assay parameters

Phosphate standard (mM)

Phosphates per lipid

Confidence level

Expected conc (mM)

Gravimetric ±%

Sample name (optional)

Compute standard concentration from mg/mL

Standard (mg/mL)

Formula weight (g/mol)

Note: 1 mM = 1 nmol phosphate per µL of standard, so nmol Pi in each standard tube = (standard volume in µL) × (standard mM).

Plan my assay — design the standard curve before you start

Standard curve size:

Why this many standards?

Choosing the number of standards is a balance, not a fixed rule.

More standards help two ways. Each one adds a degree of freedom to the curve's error estimate, which lowers the 95% t-multiplier — steeply at first, then flattening — and the 1/n term in the calibration uncertainty shrinks. You also need enough distinct levels to confirm the response is linear and to survive discarding one bad standard.

standards	df	95% t
8 (compact)	6	2.45
10 (standard)	8	2.31
12 (robust)	10	2.23

Moving compact → standard → robust tightens the calibration part of your final CI by roughly 14% then 26% (same curve quality assumed).

Fewer standards save real effort. Every standard is a full tube through the ~3-hour ash-and-develop cycle, and it competes with your replicate samples for space on the heat block.

Rule of thumb: compact (8) when time or tubes are tight; standard (10) for routine work; robust (12) when you want the tightest, most outlier-proof curve. Beyond ~12 the t-multiplier and 1/n have mostly flattened — better to spend the effort centering and spreading your standards, which attacks the leverage term directly.

Enter your expected concentration above (you usually know it to within a few percent from weighing out the lipid). The planner will recommend a sample volume and a standard curve that put your reading in the best-determined part of the curve — where the standard-curve contribution to your uncertainty is smallest.

Calibration standards

Include ≥2 blanks (0 µL) for the intercept. Uncheck a row to drop it from the fit.

use	Std vol (µL)	Absorbance	nmol Pi

Replicate samples (unknowns)

One row per tube. Volumes are typically multiples of 0.5 µL (25 µL Hamilton, 50 clicks).

use	Sample vol (µL)	Absorbance	status

technique: