Hplc Basic

High Performance Liquid Chromatography
Resolve Mixtures with similar analytes

HPLC
Triamcinolone Hydrocortisone

HPLC (Waters & HP1100)

Waters

HP 1100
Mobile Phase Solvent Cabinet Vacuum Degasser Pump Autosampler Column Compartment Detector
Control Module

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HPLC Procedure
1. Reading monograph 2. Choose correct column 3. Prepare Washing Column Solvent ( Strength same M. P.but no acid or buffer) & prepare Mobile Phase 4. Washing Column to condition 5. Setup method & Sequence 6. Prepare Standard solution & run method 7. Data analysis 8. Prepare sample solution & run sequence 9. Data analysis & print out report

Chromatographic Process

Chromatographic Process

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BAND
BAND: The distribution of an analyte from the sample in the chromatographicsystem. When the band passes through the detector, the result is a PEAK. In practice, the two terms are often used interchangeably

Baseline
BASELINE: the response of the detector on the data system display when no sample band is passing through the detector.
The "baseline" is the detector response when no sample is eluting from the column.

This chromatogram has 6 bands (peaks)

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BandSize
BAND WIDTH: The width of the band measured at its base. Also called BASELINE WIDTH. The "Baseline" width of a peak misses some area at the front and back!
PEAK AREA or HEIGHT Used to measure the size of the peak for use in quantitative analysis.

Peak Height and Width ht, W 0.5 & Wb

Baseline Drift
BASELINE DRIFT: a baseline that is not perfectly horizontal, but rises or falls withtime.
In the "real world", not all baselines are flat and horizontal.

Tailing Factor
TAILING FACTOR: a measure of how much a band deviates from being perfectly bell-shaped or symmetrical; calculated as shown here. The Tailing Factor is one way of describing tailing

Plate Number
PLATE NUMBER (N): a number that describes how good a column is in keeping sample bands narrow. Columns with largeplate numbers give narrow bands; long columns packed with small particles give the highest plate numbers.

Resolution
RESOLUTION (Rs ): defines how well separated two adjacent bands are. Larger values of resolution mean better separation.

For a perfectly symmetrical peak, both of these calculations should yield the same value for N

Resolution is the ratio of separation to average baselinewidth.

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Resolution Value

DEAD TIME
COLUMN DEAD-TIME (t0): The time it takes for solvent molecules or unretained sample bands (for which k'= 0) to pass through the column. “Uracil” is often used to measure t0 for reversed-phase columns.
Retention time can vary with changes in flow or column size.

Capacity Factor
CAPACITY FACTOR (k'): is one way to measure sample retention; Laterbands have k'-values that increase with band retention time. Values of k' for each band or compound are constant if experimental conditions do not change. k' does not change when flow rate or column dimensions are changed. k' can change when mobile phase composition, stationary phase chemistry, or temperature change.

Separation Factor
SEPARATION FACTOR (α): defined for two adjacent bands as theratio of k' for the second band divided by k' for the first band; when the separation factor equals 1.00, the two bands are on top of each other and completely unseparated. Changing the experimental conditions can increase α and permit the two bands to be separated. Also sometimes called RELATIVE RETENTION.

Naproxen Tablets

Naproxen Tablets ⎯ HPLC Assay
Assay— Mobile Phase — Prepare asuitable mixture of water, acetonitrile, and glacial acetic acid (49:50:1). Make adjustments if necessary (see System Suitability under Chromatography ). Increased resolution may be achieved by increasing the proportion of water in the Mobile phase.

Naproxen Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of C14H14O3

Solvent Mixture — Prepare a...