Domain: Healthcare — Pharmaceutical Chemistry
Target concept: Assay of Vitamin B1

Video generation prompt: An appropriate amount of vitamin B1 sample was placed in a test tube, followed by the addition of sodium hydroxide solution and potassium ferricyanide solution. After heating, n-butanol was added, and the image was aligned with the solution inside the test tube.

Evaluation guide:
(i) Target concept: Thiochrome fluorescence assay for vitamin B1 quantification.
Minimum mechanism: Alkaline oxidation of thiamine to thiochrome must occur *before* extraction; thiochrome must be *lipophilic* to partition into n-butanol; fluorescence requires intact thiochrome structure and is *only* observable in organic phase.
(ii)Storyline: [0–2s] White crystalline solid placed in upright test tube → [2–4s] Colorless NaOH solution added → [4–6s] Yellow-brown K3[Fe(CN)6] solution added → [6–8s] Flame applied beneath test tube (solution visibly warms, no boiling) → [8–10s] Colorless n-butanol added, test tube swirled, then held still: upper layer separates and emits blue fluorescence while lower layer remains orange-yellow; entire sequence captured from fixed frontal perspective with test tube centered and fully in frame, interior clearly visible.

Manual:
Experimental Procedure
1. Place white crystalline vitamin B1 sample in clean test tube.
2. Add colorless sodium hydroxide solution.
3. Add yellow-brown potassium ferricyanide solution.
4. Heat mixture gently until color develops.
5. Cool slightly, then add colorless n-butanol and shake.
Observation of Experimental Phenomena
After shaking and settling: two distinct layers form; blue fluorescence appears exclusively in the upper n-butanol layer under ambient/UV light.
Summary of Experimental Principles
Vitamin B1 (thiamine) under alkaline conditions is oxidized by ferricyanide to thiochrome, which is extracted into n-butanol and exhibits characteristic blue fluorescence.

Prompt Grounding rubric:
level_1: No test tube appears anywhere in the shot; no white/crystalline vitamin B1 sample is shown being placed into a container; none of sodium hydroxide solution, potassium ferricyanide solution, or n-butanol is added; no flame or heat source is shown beneath the tube; the camera view never settles on the tube's interior long enough to see any liquid.
level_2: A test tube holding a solid or powder appears, but nothing on screen identifies it as vitamin B1 (no comparable crystalline appearance, no labeled container); at most one of the three reagents (NaOH solution, K3[Fe(CN)6] solution, n-butanol) is added, or the reagents are added out of order, or poured into a different vessel than the original tube; a flame or heat source appears somewhere in the shot but is not shown positioned under the test tube; the camera never gives a clear, centered view into the tube's interior.
level_3: The test tube holds a visible white/crystalline solid; sodium hydroxide solution and potassium ferricyanide solution are each added as separate, visually distinguishable pours directly into that same tube; a flame or heat source is shown beneath the tube; n-butanol is added after the heating step; the camera holds the test tube upright and centered with a clear line of sight into the liquid inside.
level_4: All elements of 3-score are present AND: the solid is distinctly white and crystalline (matching vitamin B1 hydrochloride's appearance); the NaOH solution is colorless when added; the K3[Fe(CN)6] solution is visibly yellow-to-red-brown when added; the n-butanol is colorless when added; every addition goes into the one continuous test tube with no swap to a second vessel; heating precedes the n-butanol addition; the camera keeps the same test tube in the same framing and orientation from first reagent to last.
level_5: All elements of 4-score are present AND: the five steps occur in the correct order: solid placed, NaOH added, K3[Fe(CN)6] added, heating shown (with a visible thermal cue such as rising convection currents or condensation beading on the tube's upper walls), then n-butanol added; after the n-butanol addition and mixing, two clearly separated liquid layers are visible with a sharp, stable boundary between them (aqueous phase below, n-butanol phase above); camera framing is precise throughout: the tube's full height, the liquid boundary, and the open top are all in frame with no cropping.

Scientific and Causal Correctness rubric:
level_1: No hue change occurs after potassium ferricyanide is added and the tube is heated; no separation into two liquid layers occurs after n-butanol is added; no blue fluorescence appears anywhere in the tube at any point; the visual outcome bears no relation to the reagent that was just added (e.g., a color change appears before ferricyanide is added, or fluorescence appears before n-butanol is added).
level_2: A color shift happens somewhere in the sequence, but it does not follow the ferricyanide addition and heating step, or shows up before those reagents enter the tube; if two layers form, the colored or fluorescent layer sits at the bottom instead of the n-butanol layer floating on top; any fluorescence shown is unrelated to the heating step or to the n-butanol addition.
level_3: After potassium ferricyanide is added and the mixture is heated, the solution takes on a yellow-to-orange color; after n-butanol is added and the tube is mixed, two immiscible layers form with the paler layer on top; blue fluorescence is not required at this level, but if shown, it must appear only after the n-butanol has been added and only in the upper (n-butanol) layer, never in the lower aqueous layer.
level_4: All elements of 3-score are present AND: the lower aqueous layer is orange-yellow while the upper n-butanol layer starts out colorless before any fluorescence is triggered; once fluorescence appears (under UV light or as an inherent glow), it is confined to the n-butanol layer and is not visible in the aqueous layer beneath it; no fluorescence appears at any point before the n-butanol has been added and mixed in.
level_5: All elements of 4-score are present AND: blue fluorescence appears as soon as the n-butanol is mixed into the tube, without a long unexplained delay; the glow is even across the whole n-butanol layer rather than patchy or confined to one spot; the n-butanol layer shows no leftover orange color or cloudiness; the fluorescent glow stays confined to the n-butanol layer with no visible spillover or haze crossing into the aqueous layer below.

Spatiotemporal Consistency rubric:
level_1: The test tube vanishes from the shot partway through and a visually different tube appears in its place; the liquid inside jumps from one fill level or color to another between consecutive frames with no pouring motion shown connecting the two states; the flame beneath the tube switches on or off instantly with no visible ignition or extinguishing; the camera viewpoint snaps to a different angle or distance mid-shot with no pan or cut; the overall scene lighting flips between bright and dim with no gradual change.
level_2: The test tube stays on screen but its size or position shifts inconsistently between shots; when NaOH or K3[Fe(CN)6] solution is poured in, the liquid level rises but no pouring stream or container is shown entering the frame; the flame flickers at an unnatural, jittery rate or disappears mid-heating; when n-butanol is added, it splashes in a way that doesn't match a liquid of its thin, watery consistency; the two liquid layers appear fully formed the instant n-butanol touches the surface, with no settling or mixing motion shown.
level_3: The test tube stays fixed in the same spot in the frame throughout; the solid, the liquids, the flame, and later the n-butanol keep sensible positions relative to one another as reagents are added; each reagent pour shows a continuous downward stream entering the tube; the flame or heat source stays lit at a steady position beneath the tube for the full heating period; the n-butanol pour looks like a smooth, continuous stream; after mixing, the two layers separate gradually, with a visible settling motion rather than snapping into place.
level_4: All elements of 3-score are present AND: no hand, dropper, or pouring vessel ever appears to pass through the solid wall of the test tube; the liquid's surface stays continuous and doesn't tear or distort as new liquid is poured in; the flame's height and color stay steady rather than jumping or flaring; there is no frame-to-frame flicker or strobing in the tube, liquid, or flame; any swirling or shaking motion moves the liquid in a way consistent with a real fluid's momentum, with no instant stopping or unnatural sloshing.
level_5: All elements of 4-score are present AND: the test tube's glass shows no warping or bending near the frame edges; every motion (pouring, swirling, settling) plays out smoothly across the whole clip with no stutters or freeze-frames; the shadow cast by the flame and by the test tube itself stays anchored to the same relative position on the tube and background throughout the heating step, never jumping to a new direction between frames; the camera holds its single fixed viewpoint with no drifting or wobbling; the boundary between the n-butanol layer and the aqueous layer stays sharply visible and is never obscured by unnatural motion blur.