Medicinal plants post-mids

HPLC core idea

A liquid (the mobile phase) pushes your sample through a tube packed with solid particles (the stationary phase).
Different molecules interact differently with this solid packing, so they move at different speeds and come out at different times.
This difference in travel time allows separation.

Main components of an HLPC (solvent reservoid, pump, injector)

1. Solvent Reservoir

Holds the mobile phase (e.g., water, acetonitrile, methanol).
The solvent composition can be constant (isocratic) or gradually changed (gradient).

2. Pump

Pushes the mobile phase at very high pressure (typically 1,000–6,000 psi).
High pressure is needed because the column packing is very fine.

3. Injector

Introduces a precise, tiny volume of sample (usually 5–20 µL) into the flowing mobile phase.
This ensures reproducibility.

HPLC components (Column, detector, chromatogram)

4. Column

The “heart” of the system.
It contains tightly packed silica-based particles, often treated with different chemical groups (C18 is the most common).
Molecules interact with these particles depending on their polarity, hydrophobicity, charge, etc.

5. Detector

Measures molecules as they leave the column.
Common detectors:

• UV-Vis (most common)

• Fluorescence

• Refractive index

• Mass spectrometer (LC–MS setup)

The detector converts chemical presence into an electrical signal.

6. Chromatogram

A graph of detector response vs time.
Each peak represents a separated compound.
Retention time (t_R) helps identify compounds.
Peak area helps quantify them.

How separation happens in HPLC

Normal-phase HPLC

• Stationary phase: polar

• Mobile phase: nonpolar

• Polar molecules stick longer → longer retention times.

Reverse-phase HPLC (most widely used)

• Stationary phase: nonpolar (e.g., C18)

• Mobile phase: polar (e.g., water + acetonitrile)

• Nonpolar compounds stick longer.

Ion-exchange HPLC

Separates based on charge.

Size-exclusion HPLC

Separates based on molecular size; larger molecules elute first.

Why is high pressure needed in HLPC

2. Why High Pressure Is Needed

• The column is packed with extremely small particles (1.7–5 µm).

• Small particles give better separation but create high resistance to flow.

• Pumps apply high pressure (up to several thousand psi) to push the mobile phase through the column.

• Pressure improves resolution and reduces analysis time.

Explain differential retention (HPLC)

3. Core Principle: Differential Retention

Each compound interacts differently with:

• The stationary phase (solid packing)

• The mobile phase (solvent mixture)

Stronger interaction → slower movement → longer retention time
Weaker interaction → faster movement → shorter retention time

This selective retention is the basis of chromatographic separation.

Retention time (HPLC)

Retention time is the time between sample injection and the detection of that compound at the end of the column.

It is used to:

• Identify compounds (qualitative use)

• Compare samples across runs

• Monitor consistency in method validation

Chromatogram (HPLC)

A chromatogram is a plot of detector signal versus time.
It displays:

• Peaks representing each compound

• Retention time values

• Peak area (quantitative information)

• Peak shape and resolution

The area under the peak is proportional to the amount of that analyte.

Resolution (HPLC)

Resolution describes how well two peaks are separated.
High resolution means:

• Distinct peaks

• Accurate quantification

• No peak overlap

Resolution depends on:

• Column efficiency

• Selectivity

• Mobile-phase strength

• Particle size

• Column length

Efficiency and plate theory (HPLC)

Efficiency describes how sharply a peak appears.
It is expressed as the number of theoretical plates (N).
Higher efficiency → narrow peaks → better separation.

The Van Deemter equation explains factors affecting efficiency:

• Eddy diffusion

• Longitudinal diffusion

• Mass-transfer resistance

Smaller particle size and optimal flow rate improve efficiency.

Selectivity (HPLC)

Selectivity is the relative difference in retention between two analytes.
It is the most important factor for achieving separation.
It depends heavily on:

• Stationary-phase chemistry

• Mobile-phase composition

• pH (for ionizable compounds)

Changing selectivity is often the first step in method development.

Isocratic vs Gradient Elution (HPLC)

Isocratic

• Mobile-phase composition stays constant

• Good for simple mixtures

• Stable baseline

• Shorter equilibration time

Gradient

• Mobile-phase composition changes during the run (e.g., increasing organic percentage)

• Required for complex mixtures

• Improves separation of compounds with very different polarities

• Achieves narrow peaks even for late-eluting analytes

Quantitative vs Qualitative Use

Qualitative

• Identify compounds by retention time

• Assess purity

• Check for degradation products

Quantitative

• Use peak area to determine concentration

• Calibration curves needed

• Strong reproducibility required

HPLC (Solvent reservoris)

2.1 Solvent Reservoirs Purpose

Hold the mobile phase solvents.

Key Points

• Usually glass or solvent-resistant plastic bottles.

• Labeled A, B, C, D for multi-solvent mixing systems.

• Mobile phase must be filtered and degassed to prevent air bubbles.

• Degassing methods include vacuum suction, helium sparging, membrane degassers.

Common Solvents

• Water (HPLC grade)

• Methanol

• Acetonitrile

• Isopropanol

• Buffer solutions (phosphate, acetate, formate)

HPLC (pump)

Purpose

Pushes mobile phase through the system at high pressure.

Key Points

• Maintains accurate, steady flow rate (0.1–2.0 mL/min typically).

• Capable of producing 1000–6000 psi depending on column size and particle size.

• Must deliver pulse-free flow for stable baselines.

Types of Pumps

• Reciprocating pumps (most common)

• Syringe pumps (low pulsation, precise)

• Pneumatic pumps (older models)

HPLC (injector)

Purpose

Introduces a precise volume of sample into the mobile-phase flow.

Key Points

• Injection volume typically 5–20 µL.

• Must be highly reproducible for quantitative work.

Types

• Manual injector (rotary Rheodyne valve): operator turns a handle to inject.

• Autosampler: robotic arm injects many samples automatically.

Sample Loop

• A fixed loop (e.g., 20 µL) fills with sample before injection.

• Ensures consistent sample volume.

HPLC (Column)

Purpose

The site where separation occurs.

Key Points

• Stainless steel tube packed with silica-based or polymeric particles.

• Typical dimensions:

  • Length: 50–250 mm

  • Internal diameter: 2–4.6 mm

  • Particle size: 1.7–5 µm

Stationary Phase Types

• Reverse-phase (RP): C18, C8, phenyl

• Normal-phase: bare silica, amino, cyano

• Ion-exchange: sulfonic (cation), quaternary amine (anion)

• Size-exclusion: porous beads sorted by size

• Chiral phases: for enantiomeric separation

Guard Column

A small sacrificial column placed before the main column to protect it from particulates and degraded compounds.

HPLC (detectors)

Purpose

Detect and measure analytes as they exit the column.

Common Detectors

1. UV–Vis detector

   • Most commonly used

   • Suitable for compounds with chromophores

2. Photodiode array (PDA/DAD)

   • Measures full UV spectrum

   • Detects peak purity and identity

3. Fluorescence detector

   • High sensitivity for fluorescent compounds

4. Refractive index (RI) detector

   • Universal detector

   • Not useful for gradient elution

5. Evaporative light scattering detector (ELSD)

   • For non-volatile compounds lacking UV absorption

6. MS detector (LC-MS)

   • Extremely sensitive

   • Provides molecular weight and fragmentation

HPLC (chromatography software)

Purpose

Records the detector signal and converts it into a chromatogram.

Functions

• Peak integration

• Peak area calculation

• Calibration curve generation

• Retention time tracking

• Method control (pump settings, gradient programming, detector wavelength)

HPLC (connecting tubings and fittings)

Purpose

Carry mobile phase and sample between modules without leaks.

Key Features

• Stainless steel or PEEK tubing

• Internal diameters selected to reduce dispersion

• Finger-tight fittings or high-pressure ferrules

HPLC (Waste management system)

Purpose

Collects waste solvent safely.

Key Points

• Must be compatible with organic solvents

• Includes vapor traps to reduce exposure

• Prevents pressure buildup in waste bottles

HPLC diagram 

Reverse phase HPLC

3.1 Reverse-Phase HPLC (RP-HPLC)

Most widely used form of HPLC.

Principle

• Stationary phase: nonpolar (hydrophobic), usually C18, C8, phenyl.

• Mobile phase: polar (water + methanol/acetonitrile).

• Separation occurs by hydrophobic interactions.

Behavior

• Nonpolar compounds interact strongly → elute later.

• Polar compounds interact weakly → elute earlier.

Applications

• Pharmaceuticals

• Natural products

• Metabolites

• Peptides

• Most small organic molecules

Normal phase HPLC

Opposite polarity pattern compared to reverse-phase.

Principle

• Stationary phase: polar (silica, amino, cyano).

• Mobile phase: nonpolar (hexane, chloroform, isooctane).

• Separation based on polar interactions.

Behavior

• Polar compounds interact strongly → elute late.

• Nonpolar compounds elute early.

Applications

• Lipids

• Fatty acids

• Isomers with small polarity differences

• Water-sensitive compounds

Ion exchange HPLC

Principle

Separation based on charge attraction between analytes and charged stationary groups.

Types

• Cation-exchange (negatively charged stationary phase binds cations)

• Anion-exchange (positively charged stationary phase binds anions)

Key Factors

• Buffer pH (controls ionization of analytes)

• Ionic strength (salt concentration controls elution)

Applications

• Amino acids

• Peptides

• Nucleotides

• Charged metabolites

• Water-soluble vitamins

• Protein charge variants

Size exclusion HPLC

Principle

Separation based purely on molecular size, not chemistry.

• Large molecules cannot enter pores → elute first.

• Small molecules enter pores and are delayed → elute later.

Applications

• Proteins

• Polysaccharides

• Polymer molecular-weight distribution

• Aggregation analysis (biopharmaceuticals)

HPLC: partioning principle

The fundamental mechanism is differential partitioning.

An analyte repeatedly moves:

• Into the stationary phase

• Back into the mobile phase

The fraction of time spent in each phase determines retention.

Partition Coefficient

Higher K → stronger retention → longer elution time.

Adsorption vs partition chromatography

Adsorption Chromatography

• Analytes adsorb to surface sites on the stationary phase.

• Common in normal-phase HPLC.

Partition Chromatography

• Analytes dissolve into a bonded stationary phase layer (e.g., C18).

• Dominant in reverse-phase HPLC.

HPLC: selectivity

Retention factor

HPLC: retention factor

Effects of Mobile-Phase Composition

In Reverse-Phase HPLC

• Higher organic % (more ACN or MeOH) → shorter retention.

• Higher water % → longer retention.

In Normal-Phase HPLC

• Higher polarity solvent → shorter retention.

• Lower polarity solvent → longer retention.

Role of pH in HPLC

For ionizable compounds, pH strongly influences retention.

If analyte is acidic:

• Low pH keeps it neutral → more retention in RP-HPLC.

• High pH ionizes it → less retention.

If analyte is basic:

• High pH keeps it neutral → more retention.

• Low pH protonates it → less retention.

pH also affects stationary-phase stability.

Temperature in HPLC

Increasing temperature:

• Lowers mobile-phase viscosity

• Sharpens peaks

• Reduces retention

• Improves mass transfer

Temperature must be controlled for reproducibility.

HPLC solvent properties

Polarity

• Controls retention strength.

• In reverse-phase (RP), increasing organic solvent (ACN/MeOH) decreases retention.

• In normal-phase (NP), increasing polarity of solvent decreases retention.

Viscosity

• Influences column pressure.

• Higher viscosity → higher backpressure.

• Acetonitrile has lower viscosity than methanol.

UV Cutoff

• Important for UV detection.

• Solvents must not absorb strongly at the detection wavelength.

• ACN and methanol have low UV cutoffs.

Miscibility

• Solvents must mix uniformly.

• Water–ACN–MeOH combinations are fully miscible.

• Hexane (normal-phase) is immiscible with water.

Common HPLC solvents

Water (HPLC-grade)

• Main component in RP-HPLC

• Must be low in particulates, organic impurities, and ions

Acetonitrile (ACN)

• Lower viscosity → lower pressure

• Strong elution strength in RP

• Low UV cutoff (~190 nm)

Methanol (MeOH)

• Slightly higher viscosity than ACN

• Different selectivity compared to ACN

• UV cutoff ~205 nm

Buffers in HPLC

Common Buffers

• Phosphate buffer (strongest buffering range, not MS-compatible)

• Acetate buffer

• Formate buffer (MS-compatible)

• Ammonium acetate/formate (volatile; ideal for LC-MS)

Important Buffer Guidelines

• Keep buffer concentration between 5–50 mM.

• Filters (0.22 µm) are required to prevent damage to pumps and columns.

• Avoid buffers with non-volatile salts in LC-MS.

• Ensure buffer pH is at least ±1 unit from analyte pKa for pH stability.

HPLC: Additives in mobile phase

Acids

• Formic acid (0.1–1%)

• TFA (0.05–0.1%)

• Acetic acid

Uses:

• Control pH

• Improve peak shape of basic molecules

• Suppress ionization for UV detection

Bases

• Triethylamine (TEA)

• Ammonia

Uses:

• Improve peak shape of acidic analytes

• Reduce interactions with residual silanols

Ion-Pairing Agents

• Tetrabutylammonium salts

• Sodium dodecyl sulfate

Uses:

• Allow separation of highly polar or ionic compounds

• Form neutral complexes to increase retention in RP-HPLC

HPLC: Column structure

Typical Dimensions

• Length: 50–250 mm

• Internal diameter (ID): 2.1–4.6 mm (analytical)

• Particle size: 1.7–5 µm (common), sub-2 µm for UHPLC

Materials

• Stainless steel housing for high pressure.

• PEEK or titanium used in bio-compatible systems.

• End-fittings include frits that retain the packing material but allow solvent flow.

Purpose of prep HPLC

Preparative HPLC is used when:

• You need a pure chemical for further experiments

• You need to isolate natural products from plant extracts

• You want to separate and collect drug intermediates

• You need to purify proteins, peptides, or metabolites

• You want to produce reference standards

It focuses on yield, purity, and recovery, not analytical resolution.

Diff between prep and analytical HPLC

Key components of HPLC

Large-Diameter Columns

• Internal diameter of 10–50 mm or even larger

• Packed with larger particles (10–25 µm) to reduce backpressure

• Designed for durability and large sample loads

High-Capacity Pumps

• Deliver 10–100+ mL/min

• Must handle high solvent consumption and heavy loads

Fraction Collector

• Automatically collects eluted peaks into tubes/bottles

• Triggered by detector signal or retention time

UV Detector

• Monitors when target compound is eluting

• Works at higher absorbance ranges due to larger sample loads

How prep HPLC works

1. Crude sample is dissolved in a compatible solvent.

2. Sample is injected in a relatively large volume.

3. Under high-flow, analytes separate on the column.

4. The target peak is detected with UV.

5. A fraction collector captures the desired peak.

6. Solvent is removed (rotary evaporator/freeze dryer).

7. You obtain pure isolated compound.

The purified compound can then be used for:

• Biological assays

• Structural characterization (NMR, MS, IR)

• Drug discovery

• Synthetic chemistry steps

Scaling from analytical to prep HPLC

Advatnages of prep HPLC

• Produces high-purity compounds

• Handles complex mixtures

• Scalable from mg to gram quantities

• Highly reproducible

Limitations of prep HPLC

• Expensive (solvents, large columns, high-flow pumps)

• Requires careful method optimization

• Solvent waste is very high

• Peak purity sometimes lower than analytical HPLC (must re-purify)

Gas chromatography principle

1. Basic Principle

1. The sample is vaporized in a heated inlet.

2. An inert carrier gas (e.g., helium, nitrogen, hydrogen) carries the vaporized sample through a long capillary column.

3. Compounds interact differently with the stationary phase lining the column.

4. More volatile compounds travel faster; less volatile ones spend more time interacting with the column.

5. A detector measures compounds as they elute, producing a chromatogram.

Components of gas chromatography (carrier gas supply, injector, GC column)

2.1 Carrier Gas Supply

• Helium, nitrogen, or hydrogen

• Must be pure and moisture-free

2.2 Injector / Inlet

• Heats sample to instant vaporization (typically 200–300°C)

• Split or splitless injection modes control sample amount

2.3 GC Column

Most GC systems use capillary columns:

• Long (30–60 m)

• Very narrow (0.2–0.32 mm ID)

• Coated internally with stationary phase (e.g., nonpolar PDMS, polar PEG)

Column is placed inside a temperature-controlled oven.

Components of gas chromatography (Oven, FID)

2.4 Oven

• Precisely controls temperature

• Can run isothermal or temperature programs

• Temperature programming improves separation of compounds with different boiling points

2.5 Detector

Common detectors include:

• Flame Ionization Detector (FID)

• Thermal Conductivity Detector (TCD)

• Electron-Capture Detector (ECD)

• Mass Spectrometer (GC-MS)

How separation occurs in gas chromatography

Two factors determine retention:

3.1 Volatility

More volatile → lower retention time
Less volatile → higher retention time

3.2 Interaction with Stationary Phase

• Nonpolar compounds retained on nonpolar columns by dispersion forces

• Polar compounds retained on polar columns through dipole interactions

• Column chemistry determines selectivity

Gas chromatography detectors

FID (Flame Ionization Detector)

• Most common

• Very sensitive for organic compounds

• Measures ions produced when compounds burn in a hydrogen flame

TCD (Thermal Conductivity Detector)

• Universal detector

• Measures change in thermal conductivity of carrier gas

ECD (Electron Capture Detector)

• Sensitive to halogens, nitriles, pesticides

• Common in environmental analysis

GC-MS

• Most powerful

• Provides both separation (GC) and identification (MS)

• Widely used in forensics, toxicology, and metabolite identification

Applications of gas chromatography

• Environmental pollutant analysis

• Fragrance and essential oil profiling

• Petrochemical analysis

• Forensic drug testing

• Food and flavor chemistry

Advantages of GC

• Extremely high resolution

• Fast analysis

• Highly sensitive

• Excellent for volatile compounds

• Often coupled with MS for identification

Limitations of GC

• Cannot analyze non-volatile or thermally unstable compounds

• Derivatization sometimes required to make samples volatile

• Requires dry, clean samples

GC-MS steps

2. How GC-MS Works (Step-by-Step) Step 1 — Injection

Sample is injected into the heated inlet (200–300°C), instantly vaporizing it.

Step 2 — Separation (GC part)

• An inert carrier gas (helium, hydrogen, nitrogen) pushes vapors into a long capillary column.

• Compounds separate based on:

  • Boiling point

  • Polarity

  • Interaction with stationary phase

Each compound exits the column at a different retention time.

Step 3 — Ionization (MS part)

Eluting molecules enter the mass spectrometer ion source.
Most systems use Electron Ionization (EI):

• High-energy electrons (70 eV) strike molecules

• A positively charged molecular ion forms

• Molecules fragment into characteristic pieces

Step 4 — Mass Analysis

The fragments move through a mass analyzer.
Common analyzers:

• Quadrupole

• Time-of-flight (TOF)

• Ion trap

• Orbitrap (rare in GC-MS)

Fragments separate based on mass-to-charge ratio (m/z).

Step 5 — Detection

A detector (electron multiplier) amplifies ion signal and sends it to data system.

Step 6 — Mass Spectrum

Each compound produces a unique spectrum showing:

• Molecular ion peak (M⁺)

• Fragment peaks

• Base peak (most intense peak)

This acts like a “fingerprint” for identification.

GC-MS outputs

You get two types of data:

1. Chromatogram

   • Plots signal vs time

   • Peaks = separated compounds

   • Retention times help narrow down identity

2. Mass spectrum

   • Plots ion abundance vs m/z

   • Used for exact identification

   • Compared to spectral libraries (e.g., NIST database)

GC-MS essentially gives both separation and structural identity.

GC-MS ionization method

4.1 Electron Ionization (EI) — Most Common

• 70 eV electrons

• Produces reproducible fragmentation

• Generates rich spectra

• Rough on fragile molecules (hard ionization)

4.2 Chemical Ionization (CI)

• Softer method

• Produces more intact molecular ions

• Better for determining molecular weight

Core idea of TLC

TLC separates components of a mixture based on differences in polarity and their affinity for a stationary phase vs. a mobile phase.
It is a quick, inexpensive, and qualitative technique used widely in biochemistry, organic chemistry, and analytical labs.

TLC materials

• Stationary phase: A thin layer of silica gel (SiO₂) or alumina coated on a plate (usually glass, plastic, or aluminum).
  Silica is polar and can form hydrogen bonds.

• Mobile phase: A solvent or solvent mixture (e.g., hexane:ethyl acetate).
  The solvent can be non-polar, polar, or mixed, depending on what you want to separate.

• Sample: Small drop of your dissolved mixture.

• Developing chamber: A jar with solvent at the bottom and a lid to create a saturated atmosphere.

Mechanism of TLC

TLC relies on partitioning:

• Molecules that interact strongly with the stationary phase (more polar) move slowly.

• Molecules that interact weakly with the stationary phase (less polar) move quickly with the solvent front.

Balance of:

• Adsorption onto stationary phase

• Solubility in mobile phase

Because your mixture’s components have different polarity, they move at different speeds → distinct spots appear.

TLC procedure

1. Draw a baseline in pencil (ink would dissolve).

2. Spot your sample on the baseline using a capillary tube.

3. Place plate in chamber with solvent; ensure the baseline stays above solvent level.

4. Solvent rises by capillary action and carries compounds.

5. Remove plate when solvent is near the top.

6. Mark solvent front in pencil immediately.

7. Visualize spots:

   • Under UV light (for UV-active compounds)

   • Using iodine chamber

   • Spray reagents (ninhydrin for amino acids, anisaldehyde, etc.)

Rf in TLC

TLC applications

• Checking purity of a compound

• Monitoring reaction progress (e.g., reactant disappears, product appears)

• Identifying compounds by comparing Rf values with known standards

• Selecting a good solvent system for column chromatography or preparative TLC

• Distinguishing isomers or closely related molecules

polarity in TLC

Stationary phase (silica): very polar
Mobile phase: you choose polarity

Solvent choice determines movement:

• If solvent is too polar → all spots go up too far (Rf too high)

• If solvent is too non-polar → nothing moves (Rf ~0)

• Best solvent: gives good separation (spots spaced out).

Advantages of TLC

• Fast (few minutes)

• Uses little solvent

• Cheap plates and equipment

• Can run many samples side-by-side

• Easy visualization

Limitations of TLC

• Mostly qualitative, not quantitative

• Low resolution compared to HPLC

• Not automated

• Not good for volatile samples

• Hard to scale beyond preparative TLC