EXP 4: ASSAY OF ACETAMINOPHEN TABLETS USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

CHROMATOGRAPHY

• Separation technique in which the components of a sample are distributed between the two phases.

The separation occurs due to the

components that would react differently on distinct phases. 

STATIONARY PHASE

- layer or coating on the supporting medium which interacts with the analytes.

MOBILE PHASE

- carries the solute across the stationary phase

Normal

Polar compounds are highly ADSORBED

Normal

stationary 

polar 

Normal

mobile 

non-polar 

Reverse

Non-polar compounds are highly ADSORBED

Reverse

stationary

non polar

Reverse

mobile 

polar 

ADSORPTION

• Separation involves competing interaction between adsorption at the stationary phase and dissolution in the mobile phase

• Surface binding

• Compounds are separated based on their ability to be adsorbed or to stick to your stationary phase vs. how well it would dissolve in your mobile phase

ADSORPTION

USUALLY FOR 

TLC and Normal phase HPLC

PARTITION

• Particles are separated on the components of the solvent system

• Based on the differences of solubility/partitioning between two immiscible phases (the liquid stationary phase coated with on a solid support and the mobile phase that would or is intended to contain the liquid or the gas)

PARTITION

usually for 

• reverse phase HPLC and Gas Liquid Chromatography 

ION-EXCHANGE

• Separation is based mainly on differences in the ion exchange affinities of the sample components 

• Differences in charge interaction between the analyte and the charge group on the stationary phase

ION-EXCHANGE

usually for

• separation of amino acids, proteins, and nucleotides

Paper Chromatography

• Simplest and Cheapest

• More for identification = qualitative

Thin-Layer Chromatography (TLC)

• Identification and Purity testing = qualitative but sometimes, quantitative (by calculating the rf value)

• Identification - once the mobile phase travels, we could spray some reagents. If the reagent reacts, we could identify based on the classification.

Column Chromatography

More on separation/purification = qualitative

Gas Chromatography (GC)

• Intended for analysis of residual solvent

• Sample must be volatile

• Qualitative and Quantitative

High-performance Liquid Chromatography (HPLC)

• We try to assay drug impurities and dissolutions. 

• Qualitative and Quantitative

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 

• determine whether the data would be of high resolution, accuracy, and reproducibility. 

• widely applied to pharmaceutical, biological, & food analysis.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 

stationary phase 

Solid (usually silica) 

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 

mobile phase 

• Liquid Solvent system

• Would flow under high pressure 

Isocratic

Same type of solvent 

Gradient Elution

• The technique of continuously changing the solvent composition during the chromatographic run

• speeds up analysis

• improves the resolution for rediluting compounds 

Chromatographic Column 

• “column”: includes

• stainless steel,

• lined stainless steel

• Polymeric columns,

packed with a stationary phase.

lined with stainless steel:

“If you want unwanted interactions”

polymeric columns: 

Special column for ion-exchange, gel permeation, or metal-free environment

LENGTH & INNER DIAMETER OF THE COLUMN

affects the separation, and therefore, typical dimensions are included in the individual monograph.

LENGTH & INNER DIAMETER OF THE COLUMN

• ↑ length

• = better resolution but longer analysis time = ↑ back pressure (resistance)

LENGTH & INNER DIAMETER OF THE COLUMN

↓ length =

faster analysis time but poor resolution

LENGTH & INNER DIAMETER OF THE COLUMN

• narrow inner diameter

• = ↑ efficacy & ↑ sensitivity

LENGTH & INNER DIAMETER OF THE COLUMN

• wide inner diameter =

• ↑ capacity for the sample; too many separations would take a long time

PARTICLE SIZE OF STATIONARY PHASE

• Larger particle size:

• lower resolution & pressure

PARTICLE SIZE OF STATIONARY PHASE

• Smaller particle size:

• higher efficacy & resolution

Apparatus: Liquid Chromatogram

1. Reservoir containing the mobile phase

2. Pump to force the mobile phase through the system at high pressure

3. Injector to introduce the sample into the mobile phase

4. Chromatographic column (contains stationary phase)

5. Detector

6. Data collection device

Degassing Unit

• To remove dissolved air (gases  such as oxygen) from mobile phase (b)

Solvent Delivery Pump

to deliver the mobile phase at a constant flow rate and pressure

Sample Vial

To store standard solution or sample solution

Column

To separate each compound contained in the sample

Column Oven

• To keep the temperature constant (d) 

Detector

• To detect the eluted compound(s) from the column (a)

Workstation

• The signal from the detector is processed and the chromatogram is displayed (f)

• does the data processing, which generates the chromatogram showing the peaks and corresponding compounds

Drain

• Waste compounds exit the detection

Procedure 

1. Equilibrate the column and detector with mobile phase at the specified flow rate until a constant signal is received.

2. Inject a sample through the injector or use an autosampler.

3. Begin the gradient program.

4. Record the chromatogram. 

5. Analyze as directed in the monograph.

Retention Time

for identification. More on Qualitative

Peak height

- account for the number of compounds in the sample. More of Quantification

CHROMATOGRAM

• A graphical representation of the detector response, concentration of analyte in the effluent, or other quantity used as a measure of effluent concentration versus effluent volume or time.

RETENTION TIME (t_R )

• is the time elapsed between the injection of the sample and the appearance of the maximum peak response of the eluted sample 

AREA UNDER A PEAK

• It is a measure of the concentration of the compound it represents.

analysis formula

ACCEPTANCE CRITERIA

90.0% - 110.0% on the dried basis

Solution A:

1% (v/v) glacial acetic acid in water

Solution B:

Methanol