Food Analysis Lab · W4

Manual Titration vs PASCO Automatic Titration

Manual vs Automated Titration — Titratable Acidity

Food Analysis Lab · 4-hour session
Titratable acidity · KHP standardization · titration-curve inflection point · method-comparison QC

0.1 N NaOHKHP standardizationPhenolphthalein pH 8.2PASCO real-time curveBland–Altman

Pre-lab video (2 min) (Chinese narration)

Watch before class: two drinks both at pH 3.5 — is their acidity the same?

Contents: titration curve (buffer region → half-equivalence point pH = pKa → equivalence point) → phenolphthalein vs dpH/dV to find the end point → why red wine needs potentiometric titration → from mL to % acid, and standardizing NaOH with KHP first. Open on YouTube →

Before you start, watch two factory videos

How do beverage factories watch pH and titratable acidity at the same time?

Yakult production-line QC (09:56–11:25, titratable acidity + Brix) Yogurt production-line QC (12:38–13:39, pH 4.4, titratable acidity 0.9%)

A note on the video: The Yakult video calls its 130–150 °C treatment “pasteurization,” but that temperature is actually sterilization — watch first, and we’ll come back to this wording difference in class.

Today’s question: Why does factory QC measure pH and titratable acidity together? Wouldn’t one of them do?

Today’s learning goals

After W4 you should be able to…

Carry out one manual titration (phenolphthalein end point) and one PASCO potentiometric titration, and compare the two
Use KHP to standardize the true concentration of 0.1 N NaOH, and understand why standardization is needed
Read a titration curve’s buffer region, half-equivalence point and equivalence point, and see how the first derivative finds the inflection point

Use N×V×equivalent weight/(W×1000)×100 to calculate % acid, and know which predominant acid to report it as
Use a Bland–Altman plot to check whether the manual and PASCO methods agree (not just the correlation coefficient r)
Follow NaOH/phenolphthalein safety rules and complete the 4-hour division of labor and station rotation

Theory review: the definitions and principles of pH and titratable acidity are not repeated in this lesson — the full content is in the Ch22 pH and Titratable Acidity materials.

Terms first

Titratable acidity, standardization, N, equivalent weight

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Titratable acidity (TA)

The total amount of acid neutralized by a standard base titrated to a specified end point (phenolphthalein pH 8.2), expressed as % of the predominant acid

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Standardization and primary standards

NaOH absorbs moisture and CO₂, so its concentration is uncertain; use a pure, stable, non-hygroscopic primary standard (KHP) whose moles are known from its weight to back-calculate its true concentration

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N (normality)

Moles of H⁺ that can be given/accepted per liter; NaOH and HCl exchange one H⁺ each, so N = M

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Equivalent weight

Molecular weight ÷ number of neutralizable H⁺: acetic acid 60.05, lactic acid 90.08, tartaric acid 150.09÷2=75.04, citric acid 192.12÷3=64.04

How to read a titration curve

Six terms

Equivalence point: moles of NaOH added = moles of acid in the sample (theoretically exact neutralization)
End point: the point at which completion is “seen” experimentally (phenolphthalein color change or dpH/dV peak); a good method makes the end point ≈ the equivalence point
pKa: the pH at which a weak acid is half dissociated; acetic acid 4.76
Half-equivalence point: when NaOH added is half the equivalence amount, [HA]=[A⁻], and from Henderson–Hasselbalch pH=pKa+log([A⁻]/[HA]) we get pH=pKa
Buffer region: the plateau near the half-equivalence point where pH barely changes as base is added
Inflection point = steepest part of the curve = equivalence point; the first derivative dpH/dV = how much pH rises per 1 mL added (slope), and its peak is the inflection point

Ch22 pH and Titratable Acidity ↗

Simulated curve · review

Titration-curve principle: 0.075 M acetic acid / 0.1 M NaOH

Simulated curve (computed with a Python charge-balance equation, not hand-drawn). Half-equivalence point pH=pKa≈4.76; equivalence point V≈18.75 mL, pH≈8.69; phenolphthalein pH 8.2 falls at V≈18.744 mL, only 0.006 mL from the equivalence point (less than one drop). See the Ch22 materials link for the full theory.

Simulated curve · derivative

Finding the end point in potentiometric titration: dpH/dV peak = inflection point

Same simulated data, finite differences at 0.25 mL spacing; the peak is at V≈18.62 mL with dpH/dV≈8.3, nearly coinciding with the theoretical equivalence point of 18.75 mL. On a real instrument the peak is lower and broader, but its position is still usable — the benefit is that it doesn’t depend on color, so even dark-colored samples get an objective end point.

Calibration before titrating

NaOH standardization · CO₂-free water · pH sensor calibration

Standardize NaOH with KHP: potassium hydrogen phthalate (KHP, MW 204.22, must be dried first) is pure and non-hygroscopic, an ideal primary standard acid — NaOH is strongly hygroscopic and also absorbs CO₂ from air, so every batch must be re-standardized.
Reagents and probe: prepare NaOH with CO₂-free water (boiled or sparged with nitrogen); calibrate the pH sensor in the PASCO software with pH 4/7/9 buffers (two/three points) and apply temperature compensation (electrode slope varies with temperature; the software corrects it automatically) before titrating (Nielsen §22.3.2.2: choose two buffers about 3 pH units apart that bracket the sample; the titration end point is about pH 8.2–8.7, between 7 and 9).
If the system measures volume by drop count: first calibrate “mL per drop” by the gravimetric method (same approach as W1); drop count itself is not a volume unit.
Dilute samples with CO₂-free water too: dissolved CO₂ forms carbonic acid, consuming extra NaOH and making the measured % acid too high.

Titration procedure

Manual Titration vs PASCO Automatic Titration

Manual: add 0.1 N NaOH from a burette; a persistent pink with phenolphthalein is the end point; each sample in triplicate. PASCO: motorized burette + pH probe wirelessly send the pH–volume curve to a laptop in real time and project it, so the whole class watches the curve form together.

4-hour lab schedule

Division of labor and PASCO station rotation

Time	Contents
0:00–0:40	Lecture + demo: titration-curve terms, how to read the PASCO real-time curve, phenolphthalein end point vs inflection point
0:40–1:15	Each group standardizes its own 0.1 N NaOH with KHP (manual titration; calculate the actual N)
1:15–3:45	Manual titration of 4 samples (vinegar / orange juice / yogurt / red wine) × 3 replicates; PASCO station in rotation
3:45–4:00	Export PASCO CSV; groups enter data into the summary table

PASCO station rotation (5 groups, about 30 min each): when it is a group’s turn, it runs one automatic titration of vinegar and one of red wine (about 8–12 min each plus rinsing and sample change), and the rest of the time that group does manual titration in parallel. TAs, please do a timed trial run before class — if a single titration is clearly faster than expected, let the group do one more replicate.

In-class activity

Predict–Observe–Explain

Before PASCO starts titrating, each group first sketches its predicted pH–volume curve on paper
Add a few drops of phenolphthalein to the PASCO beaker too — during titration, at the moment you see it turn pink, mark that point on the projected real-time curve
Compare this mark with the inflection point calculated by the software: do they coincide?

Vinegar (a clean weak acid): near the equivalence point the curve rises almost vertically, and the phenolphthalein color change almost coincides with the inflection point (theoretically less than one drop apart).

Red wine (many organic acids + color): the curve climbs more gently, the equivalence point is less distinct, and the red color masks the phenolphthalein pink, so the visual end point is harder to judge and seen later than the inflection point — which is exactly why dark samples need potentiometric titration.

interactive lab

The journey of one drop of NaOH: Predict → Observe → Explain

Six acts: predict the curve → equivalence point → phenolphthalein end point → slope peak → red wine → % acid; you must answer correctly to unlock the next act. Open lab in new tab ↗

Mini-game ①Manual titration vs PASCO automatic titration: where are the advantages?0 / 8

Sort 8 statements into “Manual titration” or “PASCO automatic titration.”

Safety rules

NaOH · phenolphthalein · glassware

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NaOH is corrosive

Wear goggles and gloves throughout; if it gets on skin or eyes, rinse immediately with plenty of water and tell the TA

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Rinse the burette after use

NaOH corrodes/seizes the stopcock; right after titrating, rinse the burette and stopcock thoroughly with water

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Phenolphthalein is dissolved in ethanol

It is a flammable liquid; keep away from flames, use small amounts, and cap the bottle tightly right after use

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PASCO motorized burette

Do not touch the dispensing tip or probe connector while it is running; disassemble and clean only after finishing

Mini-game ②Order the manual titration steps— / 6

Use ▲▼ to arrange the 6 steps of manual titratable-acidity titration in the correct order.

Calculation: from titrant volume to % acid

Standardize NaOH first, then calculate the sample’s % acid

N_NaOH = m(KHP)204.22 × V(L)

% acid = N × V × equivalent weightW × 1000 × 100

KHP standardization example: weigh 0.5105 g KHP, titration uses 24.85 mL NaOH
N = 0.5105 / (204.22 × 0.02485) = 0.1006 M

Vinegar example: take 2.00 mL vinegar, uses 15.1 mL of 0.1006 M NaOH (acetic acid equivalent weight 60.05)
% acid = 0.1006×15.1×60.05/(2.00×1000)×100 ≈ 4.56%

Yogurt is sampled by weight (not volume) — same formula, but W is the sample “grams” and the result is reported as % (w/w) rather than % (w/v); lactic acid equivalent weight 90.08. Citrus/red wine → citric acid (64.04)/tartaric acid (75.04); always expressed as the predominant acid.

Keep your vinegar results: this week’s manual-titration vinegar acidity data will be used as reference values when we build the NIR–PLS model in W13.

Mini-game ③Calculation challenge: standardizing NaOH concentrationUnanswered

Weigh 0.5105 g of dried KHP (MW=204.22); titrating to the end point uses 24.85 mL NaOH. Find the N (M) of the NaOH. Formula: N = m/(204.22×V_L).

Method-comparison QC · principle first

Where does a Bland–Altman plot come from? Build it step by step

White vinegarOrange juiceYogurtRed wine

Illustrative data, n = 12; the last step = the Bland–Altman plot on the next slide

Your turn: mean x =difference y =

Method-comparison QC · hands-on

Move the sliders: can r or Bland–Altman fool you?

Scatter plot (x manual, y PASCO)

Bland–Altman plot

Constant bias (PASCO adds a fixed amount)

Proportional bias (grows with concentration)

Random error SD

Funnel: error grows with concentration

Correlation rBias95% LoA

🎯 Challenge: find a setting with r ≥ 0.99 but LoA outside the tolerance.

Method-comparison QC · reading & uses

Bland–Altman: four reading steps and applications

① Set the tolerance first

Decide “how big a difference is acceptable” from the intended use or regulations before seeing the data, so you cannot loosen it afterwards.

② Check the bias

A paired t-test asks whether the mean difference is significantly ≠ 0; a constant bias can be corrected.

③ Check the LoA

Only when the whole LoA lies within the tolerance are the methods interchangeable; a wide LoA = individual differences are unstable.

④ Check the shape

Flat band = good; slope = proportional bias (check volume/concentration calibration); funnel = error grows with level (use % differences); outlier = go back to the raw records.

When to use it — “can the new method replace the old one?”

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This lab

Manual phenolphthalein end point vs potentiometric auto-titration (same when a new titrator arrives: run a BA check before using it)

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W2 recap

Infrared moisture analyser vs oven method (the NB02 difference plot is an early Bland–Altman)

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Salt: rapid vs specific

Conductivity salinity meter vs a Na⁺-specific method (low-sodium soy sauce swaps some NaCl for KCl; conductivity also counts K⁺, so the bias changes with sample type)

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QC practice

Two operators, two laboratories, old vs new instrument, rapid method vs official reference method

Note: measure each sample once with each method (replicates need special handling); differences should be roughly normal; with small n the LoA itself is imprecise. The method was proposed by Bland and Altman in 1986 (The Lancet) to compare two clinical measurement devices.

Method-comparison QC

Manual vs PASCO: Bland–Altman plot

Paired design: for the same sample, measure once with each of manual and PASCO, and plot one point per sample: x = mean of the two methods, y = difference between them
Bias = mean difference = 0.036% (illustrative data, PASCO − manual)
Limits of agreement (LoA) = bias ± 1.96×SD = [-0.099, 0.170]: 95% of between-method differences are expected to fall within it; the two methods are interchangeable only if this width is acceptable in practice (SD=0.068, n=12)
Paired t-test (tests whether the mean difference is nonzero): t=1.80 (df=11) < critical value 2.20 → no significant systematic bias; but the red wine points are clearly more scattered

Method-comparison QC (2)

High r ≠ the two methods agree

Why not look only at the correlation coefficient r? The correlation coefficient r (−1 to 1, how much the two rise and fall together) is 0.999 in this illustrative data set (nearly perfect correlation!), yet the two methods can still be biased, and agreement for red wine is clearly worse — r only looks at “whether the two move together,” not “how big the difference is or how stable it is,” which is exactly why Bland–Altman exists.

Back to the two opening questions

pH vs titratable acidity; pasteurization vs sterilization

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Why measure both pH and titratable acidity?

pH = how much free H⁺ is in the solution → determines whether microbes can grow and how strong a heat treatment is needed
Titratable acidity = the total amount of all acid that can be neutralized by base (including undissociated weak acid) → relates to sourness intensity, fermentation progress, and quality specifications
The two are not interchangeable: proteins, phosphates and organic-acid salts in food buffer pH. Two drinks with the same pH 3.5 can differ several-fold in titratable acidity, and taste different in sourness

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Pasteurization vs sterilization

Pasteurization: about 63 °C for 30 min or 72 °C for 15 s, kills pathogens and most vegetative cells; the product still needs refrigeration
Sterilization: UHT at about 135–150 °C for a few seconds, or canning at 121 °C under pressure, kills even bacterial spores → commercial sterility, shelf-stable at room temperature. The video’s 130–150 °C falls in this category
Relation to pH: low-acid foods (pH > 4.6 and a_w > 0.85) must use conditions that can destroy botulinum spores; acidic foods can use milder heating — this is one reason factories measure pH

In industry slang, UHT is also often called “ultra-high-temperature pasteurization”; in reports and regulatory documents, please use precise terms.

Mini-game ④pH and titratable acidity live quiz0 / 5

Mini-game ⑤Match the sample to its predominant acid0 / 6

Sort 6 samples into the “predominant acid” used to express their results.

Mini-game ⑥Decision challenge: choosing a titration method0 / 5

Homework

Colab NB04

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Load the PASCO CSV

Load your group’s exported raw PASCO pH–volume data

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Find the equivalence point with the derivative

Compute the first derivative; the peak position = potentiometric titration end point

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Compare with manual titration

Same sample: manual % acid vs % acid back-calculated from PASCO

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Bland–Altman + paired t-test

Plot difference vs mean and test whether the two methods have a systematic bias

Colab NB04: Open NB04 in Colab ↗ · Beginner (no coding): Excel practice workbook EX04 ⬇. Don’t forget: your manual-titration vinegar acidity data will be used for the W13 NIR–PLS model, so record and keep it carefully.

Self-check

By the end of today you should be able to…

✓Say why factories measure both pH and titratable acidity
✓Explain the titration curve’s half-equivalence point (pH=pKa), equivalence point, and that the first-derivative peak = inflection point
✓Complete the KHP standardization of NaOH and calculate the correct N
✓Say why we calibrate the pH sensor, use CO₂-free water, and calibrate mL per drop
✓Complete one manual titration and one PASCO automatic titration, and compare the results
✓Use N×V×equivalent weight/(W×1000)×100 to calculate the sample’s % acid
✓Explain why Bland–Altman is more reliable than the correlation coefficient r alone when comparing two methods
✓Follow the safety rules for NaOH/phenolphthalein

Tap to tick — make sure you really can do it.