Chromatography — TLC, column, GC, HPLC (principles)

4.8.7 · Chemistry › Spectroscopy & Analysis (Intro)

Chromatography exist kyun karta hai?

Problem: Real samples (ek plant extract, ek drug batch, exhaled breath) mixtures hote hain. Analyse ya purify karne ke liye, hume components ko physically separate karna padta hai.

Trick: Do phases ke beech ek tug-of-war set karo:

Ek stationary phase (fixed: paper, silica, ek coated tube wall) jo molecules ko pakadta hai.
Ek mobile phase (moving: solvent ya gas) jo molecules ko saath le chalti hai.

Har component lagaataar "stuck on stationary" aur "riding the mobile" ke beech hop karta rehta hai. Ek molecule apna kuch fraction of time stuck rehta hai; jitna zyada chipkta hai, utna zyada peeche rehta hai. Ek lambe path par ye chhote-chhote differences milke poori separation ban jaate hain.

Ek molecule ki speed $K$ par kaise depend karti hai? (Derivation from scratch)

Kya chahiye: mobile phase ke comparison mein ek analyte band ki velocity.

Ek molecule tabhi aage badhta hai jab wo mobile phase mein hota hai. Isliye uski average forward speed hai: $v_{analyte} = v_{mobile} \times (fraction of time in mobile phase)$

Maano ki kisi bhi instant par mobile phase mein molecules ka fraction = ek molecule wahin spend karta hai time ka fraction (ergodicity). Agar $V_{m}$ = column mein mobile phase ka volume aur $V_{s}$ = stationary phase ka volume:

Mobile mein amount $= C_{m} V_{m}$
Stationary mein amount $= C_{s} V_{s} = K C_{m} V_{s}$

$fraction in mobile = \frac{C _{m} V _{m}}{C _{m} V _{m} + K C _{m} V _{s}} = \frac{1}{1 + K ( V _{s} / V _{m} )}$

TLC — Thin Layer Chromatography

Polarity rule (normal phase silica): Silica polar hai, isliye polar compounds zyada chipakti hain → chhota $R_{f}$ ; non-polar compounds fast run karte hain → bada $R_{f}$ .

Figure — Chromatography — TLC, column, GC, HPLC (principles)

Column Chromatography (preparative, gravity)

KAISE: Mixture ko upar load karo → solvent run karo → sabse kam retained (sabse kam polar) component pehle elute (exit) hoti hai; successive fractions ko test tubes mein collect karo. Pehle baahir kyun = sabse kam sticky? Sabse chhota $k$ ⇒ sabse tez $v_{analyte}$ .

GC — Gas Chromatography

GAS kyun? Gases tezi se diffuse karte hain → bahut sharp, narrow bands → zabardast resolution. Limit: sample volatile aur thermally stable hona chahiye (ise decompose hue bina evaporate hona padta hai).

GC mein separation ka basis: mostly boiling point / volatility + stationary film ke liye affinity. Low-boiling (volatile) compounds vapour ke roop mein zyada time bitaate hain ⇒ chhota $k$ ⇒ pehle elute hote hain.

HPLC — High-Performance Liquid Chromatography

High pressure kyun? Chhote particles bahut bada surface area dete hain aur kaafi equilibration steps (great separation) lekin flow resist karte hain. Ek high-pressure pump solvent ko usable speed par andar force karta hai.

Normal phase: polar stationary (silica), non-polar mobile → polar analytes zyada retain hoti hain.
Reverse phase (common): non-polar stationary (C₁₈ chains), polar mobile (water/MeCN) → non-polar analytes zyada retain hoti hain, polar pehle elute hoti hain. (TLC rule ko reverse kar deta hai!)

GC ki jagah HPLC kyun? Non-volatile / heat-sensitive samples (proteins, sugars, drugs) ke liye tum vaporise nahi kar sakte — HPLC sabko solution mein rakhta hai.

Comparison table

	TLC	Column	GC	HPLC
Mobile phase	liquid (capillary)	liquid (gravity)	inert gas	liquid (pumped)
Stationary	silica plate	silica in column	coated capillary film	packed fine particles
Main use	quick analysis	purification	volatile mixtures	non-volatile / precise analysis
Output	$R_{f}$ spots	fractions	$t_{R}$ peaks	$t_{R}$ peaks

Common mistake Steel-manned errors

(1) " $R_{f}$ sirf compound par depend karta hai." Kyun sahi lagta hai: Ek given run mein har spot ka ek fixed $R_{f}$ hota hai. Fix: $R_{f}$ solvent polarity, plate, temperature par bhi depend karta hai. Hamesha conditions quote karo; isliye hum knowns ko saath-saath run karte hain.

(2) "Higher polarity ka matlab hamesha aage travel karna hai." Kyun sahi lagta hai: "Polar = zyada react karta hai = zyada move karta hai" active lagta hai. Fix: Polar silica par (normal phase), polar = stuck = kam travel karta hai ( $R_{f}$ chhota). Reverse-phase par flip ho jaata hai. Direction is baat par depend karta hai ki kaunsa phase polar hai.

(3) "Bada $K$ matlab pehle baahir aata hai." Kyun sahi lagta hai: "K bada" subconsciously "bada/fast" padhta hai. Fix: $K$ bada ⇒ stationary prefer karta hai ⇒ $k$ bada ⇒ $v = v_{m} / (1 + k)$ chhota ⇒ sabse baad baahir aata hai.

(4) "GC kuch bhi analyse kar sakta hai." Fix: Sirf volatile, stable samples. Sugars vaporise hone se pehle char ho jaati hain → HPLC use karo.

Recall 12-saal ke bacche ko samjhao (hidden)

Ek river ki kalpana karo jo swimmers ki ek bheed ko downstream carry kar rahi hai, lekin riverbed shehed ki tarah chipchipa hai. Kuch swimmers slippery hain aur shehed ko muskil se touch karte hain — wo aage nikal jaate hain. Kuch chipku hain aur neeche se lagaataar chipakte rehte hain — wo peeche reh jaate hain. Bhale hi sabne saath shuru kiya, river ke end tak sab alag-alag times par pahunchte hain, sab spread out aur separated. Shehed-bed hai stationary phase, river hai mobile phase, aur har swimmer kitna chipku hai, wo uska $K$ hai.

Flashcards

Kisi bhi chromatography ko define karne wale do phases kaunse hain?

Ek fixed stationary phase aur ek moving mobile phase.

Partition coefficient

K = C_{s} / C_{m}

kya measure karta hai?

Ye measure karta hai ki ek analyte mobile phase ke comparison mein stationary phase ko kitna prefer karta hai.

Retention factor

k

ke terms mein analyte velocity derive karo.

v_{ana l y t e} = v_{m o bi l e} / (1 + k)

, kyunki ek molecule tabhi move karta hai jab wo mobile phase mein ho, jiska time-fraction

1/ (1 + k)

hai.

TLC mein

R_{f}

define karo.

R_{f}

= (spot ke dwara move ki gayi distance) / (solvent front ke dwara move ki gayi distance).

Normal-phase silica par, kya ek polar compound ka high ya low

R_{f}

hota hai?

Low — wo polar silica se chipakti hai aur peeche reh jaati hai.

Column chromatography mein pehle kaunsa component elute hota hai?

Sabse kam retained (sabse chhota

k

), usually silica par sabse kam polar.

GC mein mobile phase kya hai?

Ek inert carrier gas (He ya N₂).

GC ke liye sample ki kya property zaroori hai?

Volatility aur thermal stability (decompose hue bina vaporise hona chahiye).

HPLC ko high pressure kyun chahiye?

Fine packing particles great separation dete hain lekin high flow resistance hoti hai; pressure solvent ko andar force karta hai.

Normal aur reverse-phase HPLC mein kya fark hai?

Normal: polar stationary, polar analytes retain karta hai. Reverse: non-polar (C18) stationary, non-polar analytes retain karta hai.

Bada

K

matlab compound pehle ya baad mein elute hoga?

Baad mein — bada

K

⇒ stationary prefer karta hai ⇒ dheere move karta hai.

Retention time

t_{R}

tumhe kya batata hai?

Identity (kaunsa compound) fixed conditions mein; peak area quantity deti hai.

Connections

Polarity and Intermolecular Forces — $K$ set karta hai (molecules kyun chipakti hain).
Capillary Action — TLC mein solvent rise drive karta hai.
Distribution / Partition Equilibrium — $K = C_{s} / C_{m}$ ka idea.
UV-Vis Spectroscopy — common HPLC detector.
Mass Spectrometry — GC-MS / LC-MS ke roop mein identification ke liye coupled.
Purification Techniques — column chromatography ek prep method ke roop mein.