Peak using separate injections

Under some conditions, it is difficult to incorporate an internal standard into a method. If the chromatogram is very complex, an internal standard may interfere with quantitation of a peak of interest. The development of highly precise sample transfer techniques, including modem autoinjectors, reduces the dependence of the experimentalist on the use of an internal standard to correct for effects of dilution and transfer losses. In many cases, external standardization can be used effectively. The weight percent purity is determined by comparing the area of each peak in a chromatogram with those generated by separately injected pure standards of known concentration. 

Procedure. Separately inject about 2.0 pL each of the standard preparation and the assay preparation, record the chromatograms, and measure the peak responses, for valproic acid and biphenyl peaks. Calculate the quantity, in mg, of C8H1602 in the portion of capsules taken using the following formula ... 

In 2006, International Olive Oil decided to validate a method for phenolic snbstance evalnation and the decision was to validate both a colorimetric and a HPLC method. The HPLC method is the one already validated by the Italian Technical Committee for Fats and Oils, based on the Cortesi s method. Phenolic substances are extracted by means of methanol/water 80/20 and injected on a RP C18 Spherisorb ODS-2 type colnmn, 25x0.46 cm, 5 jm particle size, and the detection is carried out at 280 nm. Syringic acid is used as the internal standard. 27 peaks are separated and identified, enclosing some oxidized forms of aglycones. 

Using this or similar mixtures, inject a sample into an equilibrated column, elute the resolved bands, and record them on the recorder. Calculate plate counts for the first and last peak using the 5 [Pg.38]

Inject separately 20 [A of solutions (1) and (2) and calculate the percentage content of vigabatrin from the areas of the peaks using the declared content of C6HnN02 in vigabatrin BPCRS. 

Procedure The assay is performed by separately injecting equal volumes (about 20 pL) of the Standard Solution and the Test Solution into the chromatograph, recording the chromatograms, and measuring the responses for the major peaks. The percentage of aspartame related compound A in the test article is calculated using ... 

Procedure Using a metal-free syringe, separately inject suitable portions (about 5 p.L) of the Standard Solution and the Test Solution into the gas chromatograph, and record the chromatograms. Measure the peak area ratio of dimethyl carbonate to that of the internal standard obtained with the Standard Solution. Similarly, measure the same peak area ratio for the Test Solution. The ratio is equal to or smaller than that obtained with the Standard Solution. 

Perform the first run on the short end of the capillary. Use an injection size of 50 millibar seconds (mbs) or 0.5 pounds per square inch seconds (psi-s), depending on which type instrument you have. Set the voltage polarity to negative. Use a voltage ramp from 0 to 30 kV over 0.3 min. If the peak does not elute within 5 min, reverse the polarity and repeat the separation. Even if a peak elutes using negative polarity, reverse the polarity and perform another run in any case. In this fashion, you can determine if any sample components are going in a direction opposite to one another. This is more likely to occur at low pH when the EOF is low or when coated capillaries are employed. 

Rieger and Ballschmiter [43] developed a low resolution method for PCA analyses, based on GC/ECNI-MS in the SIM mode. Their method of quantifying PCAs in environmental samples was based on a triangulation method previously developed in their laboratory for the analysis of toxaphene [56], For PCAs, typically four ions,known to be prominent in the external standard, were monitored in separate injections of a known amount of standard and in the sample, and the areas of the broad PCA peak were then compared. The choice of external standard used was based on pattern matching, i.e., visually comparing the elution time and signal structure of the sample to those of a number of in-house standards [57],... 

Fig. 15.8. Adsorption and elution of 100 pL of a placebo chewing gum extract spiked with nicotine (0.5 mg/mL) and /S-nicotyrine, cotinine and myosmine (0.05 mg/mL). The step elution was as follows MeCN 0-8 min, MeCN + 0.2 % TFA + 2.5% H2O 8-12 min, MeCN 12-15 min. The peak identity was established by separate injections and spectral analysis using a diode array detector. Rest refers to still not eluted -nicotyrine, cotinine and myosmine. In MISPE, only -nicotyrine could be detected in the adsorption and wash steps.

Analysis of amino acid neurotransmitters was achieved by online, precolumn derivatization with OPA. The reaction of OPA with primary amines occurs in less than 2 min. An online reactor was created by inserting a smaller capillary with the analyte into a larger capillary through, which the OPA was pumped. After the end of the smaller capillary, the analyte would be mixed with OPA and derivatized. A flow-gated interface was used for injection. The sample was swept to waste by a cross-flow buffer except when an injection was made. Then analyte was allowed to accumulate in the gap (30-50 ttm) between the reaction and separation capillaries. A small voltage, less than the separation voltage, was applied for only 200 ms to perform an electrokinetic injection. This allowed very efficient separations with only 30 ms wide peaks. 

A typical high-performance liquid chromatography (HPLC) system consists of two pumps, a mixer, an injector, a guard colunm, a separation column, a detector, an electrospray nozzle, and a mass spectrometer. The two pumps and the mixer allow establishment of desired solvent gradients. The injector is used to inject a small amount of sample into the column. In all separation techniques, definition of a small volume of injected sample is cmcial in preventing adverse broadening of peaks and a consequent loss in compmient resolution. The injected compounds are separated in the separation colunm and then detected via a simple detector or a mass spectrometer. All of the parts of the HPLC system have been miniaturized, and several research groups have been able to demonstrate on-chip HPLC separations. 

Most pharmacopoeias specify that ethambutol hydrochloride contains not less than 98.0% and not more than 100.5% of C10H24N2O2 2HCI, calculated on the dried basis. Pharmaceutical products such as tablets must contain not less than 95.0% and not more than 105.0% of the labeled amount.As for pyrazinamide, the main pharmacopoeia assay method for ethambutol is a titration method. However, an HPLC method is used for the assay of ethambutol HCl in tablets.This method requires that a liquid chromatograph equipped with a 200-nm detector and a 4.6 mm X 15 cm base-deactivated column that contains 5 p,m porous silica particles, 3-10 p,m in diameter, with chemically bonded nitrile groups (CN, LIO) is used. The mobile phase is a mixture of 1.0 ml of triethylamine and 1 L of water, adjusted with phosphoric acid to a pH of 7.0. The flow rate is about 1.0 ml/min. Separately inject equal volumes (about 50 p.1) of standard preparations and the assay preparations into the chromatograph, record the chromatograms, and measure the responses for the major peaks and calculate the quantity, in mg, of ethambutol hydrochloride present in the tablets from the peak responses obtained from the assay preparation and the standard preparation, respectively. The tailing factor must not be more than 2.0, and the relative standard deviation for replicate injections not more than 2.0%. 

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