The Chromatogram

The chromatogram can finally be used as the series of bands or zones of components or the components can be eluted successively and then detected by various means (e.g. thermal conductivity, flame ionization, electron capture detectors, or the bands can be examined chemically). If the detection is non-destructive, preparative scale chromatography can separate measurable and useful quantities of components. The final detection stage can be coupled to a mass spectrometer (GCMS) and to a computer for final identification. 

The simulated distillation method uses gas phase chromatography in conjunction with an apolar column, that is, a column where the elution of components is a function of their boiling points. The column temperature is increased at reproducible rate (programed temperature) and the area of the chromatogram is recorded as a function of elution time. 

Place 40 ml. of the mixed solvent (D) in the chromatogram cylinder, and suspend the dried strip in the closed cylinder cf. p. 223) for at least 30 minutes to allow the paper to come into complete equilibrium with the mixed solvent. Then gently lower the strip until the bottom edge dips about 5 mm. into the solvent. Allow the chromatogram to develop overnight protected from draughts. 

Purification of anthracene. Dissolve 0-3 g. of crude anthracene (usually yellowish in colour) in 160-200 ml. of hexane, and pass the solution through a column of activated alumina (1 5-2 X 8-10 cm.). Develop the chromatogram with 100 ml. of hexane. Examine the column in the hght of an ultra-violet lamp. A narrow, deep blue fluorescent zone (due to carbazole, m.p. 238°) will be seen near the top of the column. Immediately below this there is a yellow, non-fluorescent zone, due to naphthacene (m.p. 337°). The anthracene forms a broad, blue-violet fluorescent zone in the lower part of the column. Continue the development with hexane until fluorescent material commences to pass into the filtrate. Reject the first runnings which contain soluble impurities and yield a paraffin-hke substance upon evaporation. Now elute the column with hexane-benzene (1 1) until the yellow zone reaches the bottom region of the column. Upon concentration of the filtrate, pure anthracene, m.p. 215-216°, which is fluorescent in dayhght, is obtained. The experiment may be repeated several times in order to obtain a moderate quantity of material. 

Column Efficiency. Under ideal conditions the profile of a solute band resembles that given by a Gaussian distribution curve (Fig. 11.1). The efficiency of a chromatographic system is expressed by the effective plate number defined from the chromatogram of a single band. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

In capillary electrophoresis the conducting buffer is retained within a capillary tube whose inner diameter is typically 25-75 pm. Samples are injected into one end of the capillary tube. As the sample migrates through the capillary, its components separate and elute from the column at different times. The resulting electrophero-gram looks similar to the chromatograms obtained in GG or HPLG and provides... 

This experiment describes the quantitative analysis of the asthma medication Quadrinal for the active ingredients theophylline, salicylic acid, phenobarbital, ephedrine HGl, and potassium iodide. Separations are carried out using a Gi8 column with a mobile phase of 19% v/v acetonitrile, 80% v/v water, and 1% acetic acid. A small amount of triethylamine (0.03% v/v) is included to ensure the elution of ephedrine HGl. A UV detector set to 254 nm is used to record the chromatogram. 

R-propranolol), Tenormim (oL-atenolol) and Lopressor (oL-metaprolol). The mobile phase was 90 10 (v/v) acetonitrile and water. A UV detector set to 254 nm is used to obtain the chromatogram. 

In this experiment students analyze an artificial RNA digest consisting of cytidine, uridine, thymidine, guanosine, and adenosine using a Cjg column and a mobile phase of 0.4% v/v triethylammonium acetate, 5% v/v methanol, and 94.6% v/v water. The chromatogram is recorded using a UV detector at a wavelength of 254 nm. 

Using the chromatogram shown here, which was obtained on a 2-m column, determine values for t w, 4, k N, and H. 

The chromatogram in Problem 4 was obtained on a 2-m column with a column dead time of 50 s. How long a column is needed to achieve a resolution of 1.5 What height of a theoretical plate is needed to achieve a resolution of 1.5 without increasing the length of the column ... 

Alltech and Associates (Deerfield, IL) graciously provided permission to use the chromatograms in Ghapter 12 the assistance of Jim Anderson, Vice-President, and Julia Poncher, Publications Director, is greatly appreciated. Fred Soster and Marilyn Guller, both of DePauw University, provided assistance with some of the photographs. 

Paper chromatograms were developed for 50 50 blends of nylon-6,6 and nylon-6,10 after the mixtures had been heated at 290 C for various periods of time. The following observationsf describe the chromatograms after the indicated times of heating ... 

The basic premise of this method is that the magnitude of the detector output, as measured by hj for a particular fraction, is proportional to the weight of that component in the sample. In this sense the chromatogram itself presents a kind of picture of the molecular weight distribution. The following column entries provide additional quantification of this distribution, however. 

Further, peak overlap results in nonlinear detector response vs concentration. Therefore, some other detection method must be used in conjunction with either of these types of detection. Nevertheless, as can be seen from Figure Ilf, chiroptical detection can be advantageous if there is considerable overlap of the two peaks. In this case, chiroptical detection may reveal that the lea ding and tailing edges of the peak are enantiomerically enriched which may not be apparent from the chromatogram obtained with nonchiroptical detection (Fig. He). 

Development of the Chromatogram. The term development describes the process of performing a chromatographic separation. There are several ways in which separation may be made to occur, eg, frontal, displacement, and elution chromatography. Frontal chromatography uses a large quantity of sample and is usually unsuited to analytical procedures. In displacement and elution chromatography, much smaller amounts of material are used. 

Specifications, Analysis, and Toxicity. Dicyandiamide is identified quaHtatively by paper chromatography and quantitatively by ultraviolet spectrometry of the chromatogram. More commonly, total nitrogen analysis is used as a purity control or the dicyandiamide is converted by hydrolysis to guanylurea, which is determined gravimetrically as the nickel salt (50). Methods based on the precipitation of silver dicyandiamide picrate are sometimes used (51). Dicyandiamide can also be titrated with tetrabutylammonium hydroxide ia pyridine solution. Table 4 gives a typical analysis of a commercial sample. Dicyandiamide is essentially nontoxic. It may, however, cause dermatitis. 

With eluent MeOH H O = 40 60% w (flow rate -1 ml/min) full sepai ation was achieved within acetylated and non-acetylated on amino group of eight thioesters of 4-aminobenzenthiosulfinic acid with retention in next order for R - = -CH3, -C,H3,-CH,-CH=CH, -C3H3by RP-HPLC on Spherisorb-ODS-2 (250x4,6 mm). The chromatograms were obtained at 254 and 289 nm. Retention was generalized by In = In - S-(p (cp - MeOH volume pai t in range near 0.4-0.6) as shown in Fig. 

The purity of the product was determined by the checkers by GLC analysis using the following column and conditions 3-nm by 1.8-m column, 5% free fatty acid phase (FFAP) on acid-washed chromosorb W (60-80 mesh) treated with dimethyldichlorosilane, 90 C (1 min) then 90 to 200 C (15°C per rain). The chromatogram showed a major peak for methyl 2-methyl-l-cyclohexene-l-carboxylate preceded by two minor peaks for methyl 1-cyclohexene-l-carboxylate and l-acetyl-2-methylcyclohexene. The areas of the two impurity peaks were 5-6% and 0.5-2% that of the major peak. The purity of the product seems to depend upon careful temperature control during the reaction. The total amount of the two impurities was 14-21% in runs conducted at about -15 to -20°C or at temperatures below -23°C. 

Purified by chromatography on a column of deactivated alumina or magnesium oxide, or on a thin layer of silica gel G (Merck), using dichloromcthane/dicthyl ether (9 1) to develop the chromatogram. Stored in the dark and in an inert atmosphere at -20 . 

Separated from retinol by column chromatography on water-deactivated alumina with hexane containing a very small percentage of acetone. Also chromatographed on TLC silica gel G, using pet ether/isopropyl ether/acetic acid/water (180 20 2 5) or pet ether/acetonitrile/acetic acid/water (190 10 1 15) to develop the chromatogram. Then recrystd from propylene at low temperature. 

The product may be analyzed by gas chromatography on an 8 mm. x21S cm. column heated to 220-240° and packed with Dow-Corning Silicone Fluid No. 550 suspended on 50-80 mesh ground firebrick. The chromatogram obtained with this column exhibits a single major peak. The ultraviolet spectrum of an ethanol solution of the product has a maxium at 250 m>i (s = 17,200). 

The baseline is any part of the chromatogram where only mobile phase is emerging from the column. 

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