HPLC systems washing

Milbemectin consists of two active ingredients, M.A3 and M.A4. Milbemectin is extracted from plant materials and soils with methanol-water (7 3, v/v). After centrifugation, the extracts obtained are diluted to volume with the extraction solvent in a volumetric flask. Aliquots of the extracts are transferred on to a previously conditioned Cl8 solid-phase extraction (SPE) column. Milbemectin is eluted with methanol after washing the column with aqueous methanol. The eluate is evaporated to dryness and the residual milbemectin is converted to fluorescent anhydride derivatives after treatment with trifluoroacetic anhydride in 0.5 M triethylamine in benzene solution. The anhydride derivatives of M.A3 and M.A4 possess fluorescent sensitivity. The derivatized samples are dissolved in methanol and injected into a high-performance liquid chromatography (HPLC) system equipped with a fluorescence detector for quantitative determination. 

Most manufacturers of dissolution testing devices offer semi-automated systems that can perform sampling, filtration, and UV reading or data collection. These systems automate only a single test at a time. Fully automated systems typically automate entire processes including media preparation, media dispensing, tablet or capsule drop, sample removal, filtration, sample collection or analysis (via direct connection to spectrophotometers or HPLCs), and wash cycles. A fully automated system allows automatic performance of a series of tests to fully utilize unused night and weekend instrument availability. 

These columns offer the potential for creating a hybrid-silica monolith, which can be run on existing HPLC systems at high flow rates, that are temperature and pH resistant. By their very nature, these columns would be void free and the only column killers that they would suffer from would be particulates and bound organics. They probably could be reverse flushed for particulate wash out and bound materials could be washed off with strong solvents. 

Pacification—Treatment of a column-bridged HPLC system with 20% (6 N) nitric acid to remove buffer and organic deposits and protect metal surfaces from corrosion. The column must be removed before acid treatment. Overnight water wash is needed to remove the last traces of acid. 

Check your HPLC system manual to make sure the system is compatible with nitric acid washing. Make sure the reservoir sinker is made of stainless steel and not monel metal. Replace the water in the solvent reservoir with 20% nitric acid. Stop Note Make sure the column has been replaced with the column bridge. Do not pump nitric acid through a bonded-phase column. Wash the system for 15 min at 2 mL/min with 6 N (20%) nitric acid. Discard the wash carefully. 

Pacification of a columnless HPLC with nitric acid Observe the length of time necessary to wash all the nitric acid out of the HPLC system. 

Separation of enantiomers of etodolac using two different derivitization agents and three chiral stationary phases has been studied [24]. Etodolac was converted to its anilide derivative with either 1,3-dicyclohexyl-carbodiimide or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Etodolac, derivatizing agent, aniline, and dichloromethane were allowed to incubate for 30 minutes, which was followed by addition of 1 M HC1. The organic layer was removed, washed, dried, and then injected into normal phase or reverse phase HPLC. The HPLC system consisted of a 250 x 4.6 mm (5 pm particle size) column packed with chiral stationary phases, and detection was effected by the UV absorbances at 254 and 280 nm. Separation of etodolac enantiomers was achieved on only one of the stationary phases when using 20% 2-propanol in hexane as the mobile phase at a flow rate of 2.0 mL/min. 

Holtzapple et al. developed an immunoassay method for determination of four fluoroquinolone compounds (including ciprofloxacin) in liver extracts [64]. In this method, an immunoaffinity capture SPE column was used, that contained anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix compounds had been washed away, the bound ciprofloxacin was eluted directly onto the HPLC column. The HPLC system used a 5 pm Inertsil phenyl column (15 cm x 4.6 mm i.d.), with 0.1 M-glycine hydrochloride/acetonitrile (17 3) as the mobile phase (eluted at a rate of 0.7 mL/min). Fluorimetric detection at 444 nm was used after excitation at 280 nm. The recovery of ciprofloxacin ranged from 85.7 to 93.5%, and the detection limit was... 

Also of importance to note with the PVDF method, we observed Triton to contaminate the HPLC system, most notably the flow cell, after multiple injections. In several instances, this was serious enough to produce many high UV-absorbing peaks and prevented collection of the in-progress map. Periodic, extensive washing of the system may help resolve this. 

Insertion/introduction of the needle into the GC port, depression of the plunger, and thermal desorption of the analytes. Alternatively, the analytes are washed out of the fiber by the HPLC mobile phase via a modified HPLC six-port injection valve and a desorption chamber that replaces the injection loop in the HPLC system. The SPME fiber is introduced into the desorption chamber, under ambient pressure, when the injection valve is in the load position. The SPME-HPLC interface enables mobile phase to contact the SPME fiber, remove the adsorbed analytes, and deliver them to the separation column. Analytes can be removed via a stream of mobile phase (dynamic desorption) or, when the analytes are more strongly adsorbed to the fiber, the fiber can be soaked in mobile phase or another stronger solvent for a specific period of time (e.g., 1 min) before the material is injected onto the column (static desorption) (Fig. 6). 

SPE cartridges were conditioned with 2 mL each of acetonitrile and water. Derivatized sample was applied to SPE cartridge, which was washed with 400 fiL of acetonitrile-water (1 1, v/v) followed by 1600 /rL of acetonitrile-sodium acetate solution (1 1, v/v). After elution with 400 fih of methanol-conc.HCl (99 1, v/v), the eluate was allowed to dry with N2 gas. The residue was reconstituted with 100 fiL of mobile phase A, and 20 fiL of the resultant were injected into the HPLC system. 

The high salt concentrations used can present a challenge for the HPLC equipment. Pumps with seal-wash are prrferred. If chloride buffers are used, HPLC systems with nonmetallic fluid paths are recommended. It is also advisable to flush the salt solution out of the system when it is not in use. Temperature and pH can affect the separation and should therdbre be controlled. But both the temperature effect and pH effect are smaller than in other forms of chromatography (with the exception of size-exclusion chromatography). 

Polymer particles are suspended in water (25% MeCN) and then slurry-packed into stainless steel columns (250 mm x 4.6 mm i.d.) using an air-driven fluid pump (Haskel, Burbank, CA, USA) and water (25% MeCN) as the packing solvent. The packed columns are washed on-line on a Beckman HPLC system (comprising a solvent module 126 and diode array detector 168) using MeCN (20% acetic acid) to remove the print molecule until a stable base line is obtained. The mobile phase is then changed to a citrate buflfer (pH 3.0, 25 mM citrate) containing 10% MeCN (v/v) at a flow rate of 1 ml. min . For a test of chiral resolution, a racemic mixture of (+)- and ( )-isoproterenol (20 pL at 2 mM in the mobile phase) is injected, and the elution monitored at 280 nm. Acetone can be used as a void marker for the calculation of capacitor factor (k ) and separation factor (oc). 

The piecolum is washed with the buffer and is backflushed with the mobile-phase transferring the analyte to the HPLC system... 

---