Question

YOU MUST CONFIGURE AN HPLC SYSTEM THAT IS CAPABLE OF SEPARATING THE ANALYTES, DETECTING THE ANALYTES, DETERMINING THE MOLECULAR WEIGHTS OF THE ANALYTES, AND ESTIMATING THE AMOUNTS OF THE ANALYTES IN EACH SAMPLE MIXTURE. YOU MUST SELECT FROM THE FOLLOWING OPTIONS BELOW

1.PUMPS: LOW PRESSURE MIXING OR HIGH PRESSURE MIXING

2.COLUMN STATIONARY PHASE

3.COLUMN TEMPERATURE CONTROL YES OR NO

4.ISOCRATIC OR GRADIENT SEPARATION

5.MOBILE PHASE COMPOSITION

6.SEPARATION CONDITIONS-FLOW RATE, RUN TIME, GRADIENT TIME (IF USED)

7.DETECTOR(S) TO BE USED

8.METHOD FOR ESTIMATING MOLECULAR WEIGHT AND AMOUNT OF EACH ANALYTE

THEN, EXPLAIN YOUR RATIONALE FOR MAKING YOUR SELECTIONS

Answer 1

1. PUMPS: LOW PRESSURE MIXING

A liquid chromatograph for HPLC utilizes low pressure solvent metering pumps for solvent mixing in series with a high pressure pump driving the column.

RATIONALE FOR MAKING SELECTION

Low-pressure systems are capable of delivering a combination of up to 4 different solvents.

2. COLUMN STATIONARY PHASE : C18 COLUMN, SILICA

HPLC columns are usually packed with pellicular or porous particles. Silica is the most common type of porous particle packing material. The material filled in the HPLC columns is known as a stationary phase. C 18 column is specific for analysis of different products.

RATIONALE FOR MAKING SELECTION

Silica is is sued as a packing material for many reasons, such as mechanical strength, high surface area, and easily tailored pore size distributions. Its particle size and porosity that helps in separation of components and silica gel is also an inert material that does not react with mobile phases.

3. COLUMN TEMPERATURE CONTROL - YES

Accurate temperature control of your HPLC columns will improve the reproducibility of results.

RATIONALE FOR MAKING SELECTION

Operating at increased temperatures provides better separation performance and reduced analysis time.

4. ISOCRATIC OR GRADIENT SEPARATION

Isocratic elution is commonly used in HPLC

RATIONALE FOR MAKING SELECTION

Isocratic elution is typically effective in the separation of sample components that are very different in their affinity for the stationary phase.

5. MOBILE PHASE COMPOSITION

Mobile phases acetonitrile : water

RATIONALE FOR MAKING SELECTION

Acetonitrile is miscible with water and a range of organic solvents. It lower the noise in UV detection.

6. SEPARATION CONDITIONS - FLOW RATE, RUN TIME, GRADIENT TIME

flow rate 1 mL min−1  Run time 10-60 minutes gradient time 5–95% B over 10 or 20 min

RATIONALE FOR MAKING SELECTION

The main purpose of gradient elution is to move strongly retained components of the mixture faster, but having the least retained component well resolved.

7. DETECTOR(S) TO BE USED - UV

The UV, VIS, and PDA detectors are categorized as absorbance detectors. UV detector is a very commonly used detector for HPLC analysis.

RATIONALE FOR MAKING SELECTION

UV detector provide good sensitivity for light-absorbing compounds.

8. METHOD FOR ESTIMATING MOLECULAR WEIGHT AND AMOUNT OF EACH ANALYTE

Molecular weight determination by precipitation/redissolution HPLC is a relative method. So it must be calibrated. The calibration is performed by determining retention times of polymer standards with narrow molecular weight distribution.

Concentration of sample= Area of sample/ Area of standard x concentration of standard

RATIONALE FOR MAKING SELECTION

HPLC is a technique in analytical chemistry used to separate, identify and quantify each component in a mixture because of its accuracy, good separation of large molecule and determine approximate molecular weight.