Lab 12 - Ceutics exam 5

Obj. 1

Understand the basic principle of High Performance Liquid Chromatography (HPLC).

🔹Q: What is HPLC?

High Performance Liquid Chromatography (HPLC) = an analytical technique used to separate, identify, and quantify compounds in a mixture.

What is the basic principle of HPLC?

HPLC SEPARATES compounds based on their different affinities for:

• the stationary phase (column packing material)

• and the mobile phase (liquid solvent)

How does separation occur in HPLC?

A sample is injected into a column filled with stationary phase while a liquid mobile phase is pumped through at high pressure.

⭐️Different compounds interact differently with the stationary phase, causing them to travel @ different speeds and elute @ different times.

What is the stationary phase in HPLC?

The stationary phase is the porous material packed inside the HPLC column that interacts with analytes during separation.

What is the mobile phase in HPLC?

The mobile phase is the liquid solvent pumped through the HPLC column @ ⬆high pressure.

Why do compounds elute @ different times in HPLC?

• ELUTE = “to come out of the column” during chromatography.

Because compounds have different affinities/interactions with the stationary phase → causing different retention times.

What is retention time in HPLC?

Retention time is the time it takes for a compound to travel through the column and reach the detector.

What does the detector do in HPLC?

The detector converts separated compounds → into measurable signals for analysis and quantification.

What is the most common HPLC detection method?

UV-Vis absorption detection.

Why is HPLC important in pharmacy?

HPLC is used for:

• purity testing

• drug strength assays

• stability studies

• impurity detection

• quality control

How does HPLC quantify an API in a tablet?

The detector measures the API peak area/intensity and COMPARES it to a standard solution.

Simple explanation of HPLC?

HPLC pushes a liquid mixture through a special column so compounds separate and come out at different times.

🧠HPLC = high-pressure separation of compounds in liquid form.

🧠Different interactions → different retention times.

Obj. 2

Understand the primary HPLC separation modes and detection methods.

🔹Q: What determines HPLC separation modes?

HPLC separation modes are based on the type of stationary phase used in the column.

What is Normal-Phase HPLC?

Uses:

• a POLAR stationary phase

• a NONPOLAR mobile phase

⭐️LESS polar compounds elute first, while more polar compounds elute later.

What is Reversed-Phase HPLC?

Uses:

• a NONPOLAR stationary phase (ex: C18)

• a POLAR mobile phase

⭐️MORE polar compounds elute first, while nonpolar compounds elute later.

Which HPLC separation mode is most commonly used?

Reversed-phase HPLC.

What is HILIC chromatography?

Uses:

• a POLAR stationary phase

• a high-organic mobile phase

⭐️It is useful for ⬆HIGHLY POLAR compounds.

What is Ion-Exchange HPLC?

Separates compounds based on charge using ionic stationary phases.

What is Size-Exclusion HPLC?

Separates molecules based on size:

• LARGER molecules elute FIRST

• smaller molecules elute later

What is Chiral HPLC?

Separates enantiomers using a chiral stationary phase.

What is Affinity Chromatography?

Uses highly specific “lock-and-key” interactions such as antibody-antigen binding.

What is the role of HPLC detection methods?

Detection methods convert separated compounds → into measurable signals for quantification.

What is the most common HPLC detection method?

UV-Vis absorption detection.

What type of molecules are detected by UV-Vis absorption?

Molecules containing at least one double or triple bond.

What is fluorescence detection?

A highly sensitive detection method used for fluorescent molecules or conjugates.

What is refractive index detection used for?

Used for molecules with little/no UV absorption such as:

• sugars

• alkanes

What is mass spectrometry detection in HPLC?

Detects compounds by MASS analysis. When coupled with HPLC, it is called LC-MS.

What is electrochemical detection used for?

Used for electrochemically active compounds such as:

• neurotransmitters

• catecholamines

• phenols

What is Evaporative Light Scattering (ELS) Detection in HPLC?

Used mainly for NON-volatile compounds that do NOT absorb UV-Vis light well.

How does Evaporative Light Scattering detection work?

The mobile phase is evaporated, leaving analyte particles behind. Light is scattered by the particles and detected.

What type of compounds are commonly analyzed with evaporative light scattering detection?

Non-volatile, non-UV-absorbing molecules.

How sensitive is Evaporative Light Scattering detection?

• ⬆MORE sensitive than RI detection.

• ⬇LESS sensitive than UV-VIS detection.

What is radioactivity detection in HPLC?

A detection method used for compounds containing radionuclides (radioactive isotopes).

What type of compounds use radioactivity detection?

Radiopharmaceuticals and radioactive compounds.

How sensitive is radioactivity detection?

It is the MOST SENSITIVE HPLC detection method.

Simple explanation of radioactivity detection?

The detector measures radiation emitted from radioactive compounds.

🧠Easy memory trick for HPLC separation modes?

• Normal phase = polar column

• Reverse phase = nonpolar column

• Ion exchange = charge

• Size exclusion = size

• Chiral = enantiomers

🧠Easy memory trick for HPLC detection methods?

• UV = most common

• Fluorescence = very sensitive

• RI = sugars/non-UV compounds

• MS = mass analysis

• Electrochemical = redox-active compounds