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Fast Analysis of a Complex Drug Delivery System

Most pharmaceutical QC laboratories are faced with rapidly increasing demands for high throughput capability. A good example of one laboratory's

approach to addressing a large number of samples for analysis on a daily basis is summarized below.This novel drug delivery system contains a small protein and other components that span an order of magnitude in molecular weight. All the compounds of interest were easily resolved in a single analysis with a fast linear gradient on a 5cm long HAISIL 300 C18 5µm column.

Very High Peak Capacity

Depending upon the type of information a protein chemist is seeking, the HPLC analysis of a complex digest can take 1 to 2 hours.The following chromatogram

illustrates a high resolution tryptic digest map of BSA on a 250x2.1mm HAISIL 300 C18 5µm column indicates peak capacities in excess of 100. Small proteins, on the other hand can yield fewer fragments, and if only a "finger-print" of a digest is required, high peak capacity columns can be a waste of time.

HA I S I L 3 0 0 A P P L I C AT I ONS

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Tryptic Map of BSA

on HAISIL 300 C18 5µm 250x4.6mm P/N HS-2546-W185

Peptide Map in 5 minutes

The figure below illustrates a very fast

peptide map of cytochrome-C.The information that can be gleaned from this five minute profile is more than adequate for many analytical requirements.The robust 50x4.6mm HAISIL 300 C18 column provides hundreds of runs without performance loss even under the aggressive 6ml/min flow conditions used in this example.

The cytochrome-C is a good example of pushing a column beyond its optimum operating conditions (more than six times optimum linear velocity) and still being able to get desired results.The same column can be run under more conventional conditions with a flatter gradient to optain even higher resolution separations.The rhGH tryptic map below was done on a 5cm long column that replaced the conventional 25cm column that was used initially.

The Role of Column Length in Gradient Separations

Changes in column length have a direct and linear effect on theoretical plates and analysis time in isocratic separations. Column length plays a less impor tant role, however, in gradient techniques.The three chromatograms below illustrate how well packed shor t HPLC columns yield very similar peptide separations under identical gradient conditions.

For this study, the 2.1mm I.D. HAISIL 300 C18 5µm columns ranged from 250 to 100mm in length.While solvent and analysis time savings may be minimal, column cost savings can be significant when using as shor t a column as possible. Shor t columns also exhibit lower pressure at high flow rates.

250x2.1mm HAISIL 300 C18 5µm HS-2521-W185

150x2.1mm HAISIL 300 C18 5µm HS-1521-W185

100x2.1mm HAISIL 300 C18 5µm HS-1021-W185

1. Gly-Tyr 2.Val-Tyr-Val 3. Met Enkephalin 4. Leu Enkephalin 5. Angiotensin II

Fast Tryptic Map of rhGH

50x4.6mm HAISIL 300 C18