Methanol chromatograms

This experiment focuses on developing an HPLG separation capable of distinguishing acetylsalicylic acid, paracetamol, salicylamide, caffeine, and phenacetin. A Gjg column and UV detection are used to obtain chromatograms. Solvent parameters used to optimize the separation include the pH of the buffered aqueous mobile phase, the %v/v methanol added to the aqueous mobile phase, and the use of tetrabutylammonium phosphate as an ion-pairing reagent. 

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. 

Aroclor 1248, Aroclor 1254, and Aroclor 1260. Quantitation is by comparison of chromatograms with standard concentrations of pure compounds treated in an identical manner. The phenoxy acid herbicides (2,4-dichlorophenoxy)acetic acid (2,4-D), sUvex, and (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T) can be deterrnined by electron-capture detection after extraction and conversion to the methyl esters with BF.-methanol. The water sample must be acidified to pH <2 prior to extraction with chloroform. 

The most common chromatogram in the distilled spirits industry is the fusel oil content. This consists of / -propyl alcohol, isobutyl alcohol, and isoamyl alcohol. Other common peaks are ethyl acetate, acetaldehyde, and methanol. The gc columns may be steel, copper, or glass packed column or capillary columns. Additional analyses include deterrninations of esters, total acids, fixed acids, volatile acids, soHds or extracts (used to determine... 

Fig. 38 Companson of manual dipping (A) with mechanized dipping (B) on the basis of scans and calibration curves [114] — 1 = cM-diethylstilbestrol, 2 = traw-diethylstilbestrol, 3 = ethinylestradiol Scanning curve 2 ng of each substance per chromatogram zone = 313 nm, /n > 390 nm Dipping solution water — sulfuric acid — methanol (85 + 15 + 1)...

Fig. 1 Fluorescence plot (A) of the chromatogram track of an unpurified extract of sinapis seed (application 2 pi of a 1% solution in methanol) and (B) of a reference track with 1 pg sinigrin per chromatogram zone.

Note The reagent can be employed on silica gel, alumina, polyamide and cellulose layers. In the case of the latter it is to be recommended that the solutions be diluted 1+3 with methanol. The detection limit is reported to be 0.1 to 0.5 pg per chromatogram zone [5]. 

Detection and result The developed chromatogram was heated to 80°C for 15 min the warm plate was sprayed first with Flavone Reagent (3% in methanol)... 

Note The pre- and post-treatment of the chromatograms with the basic tri-ethylamine solution, which can be replaced by an alcoholic solution of sodium hydroxide [1,4] or a phosphate buffer solution pH = 8.0 (c = 0.2 mol/1) [5], serves to stabilize the fluorescence of the amino derivatives [2]. A final spraying with methanolic hydrochloric acid (chci = 5 mol/1) or 70% perchloric acid renders the detection reaction highly specific for histamine [4] and for catecholamines and indolamines [5]. 

Fig. 1 Reflectance scan of chromatogram tracks (A layer prewashed with mobile phase, with methanol) with 750 ng each substance per chromatogram zone. Sphingomyelin (1)] lecithin (2).

FIGURE 13.1 Initial Chromatograms were obtained using THF as mobile phase. Each column was then stressed by an abrupt change of mobile phase from THF to methanol. Methanol was run through each column for approximately 16 hours, at which time the mobile phase was changed back to THF. 

Comparison of the separation efficiency between two columns in the same mobile phase or one column in two mobile phases is based on the extent of resolution of the peaks of the PEO standards in the respective chromatograms of the PEO A, B, and C group. Due to the limitation of space, only the TSK PEO A chromatograms for the four columns in water and water/methanol are... 

FIGURE 4.22 HPLC chromatogram of amino acids employing precolumn derivatiza-tion with OPA. Chromatography was carried out on an Ultrasphere ODS column using a complex tetrahydrofuran methanol 0.05 M sodium acetate (pH 5.9) 1 19 80 to methanol 0.05 M sodium acetate (pH 5.9) 4 1 gradient at a flow rate of 1.7 mL/min. 

Figure 1.2 Chromatogram of coal-tar oil obtained by using the following conditions column, Waters Spherisorb PAH 5 mm in 250 p.m id X 30 cm fused silica column oven temperature, 100°C UV detector wavelength to 254 nm mobile phase, 100 to 300 bar CO2 and 0.10 to 1.00 p.L min methanol over 30 minutes.

Fig. 2-7. Chromatograms of albuterol in the new polar organie phase on vaneomyein (A), teieoplanin (B), ristoeetin A (C), vaneomyein + teieoplanin (D), ristoeetin A + vaneomyein (E), and ristoeetin A + vaneomyein + teieoplanin (F). All eolumns were 100 x 4.6 mm. The numbers by the peaks refer to the retention time in minutes. The mobile phase was methanol with 0.02 % glaeial aeetie aeid and 0.01 % triethylamine (v/v). The flow rate was 2.0 mL min at ambient temperature (23 °C).

N,N -Diacetylkasuganobiosamine. To a suspension of kasuganobiosamine (500 mg., 1.6 mmoles) in 10 ml. of methanol was added 2.5 ml. of acetic anhydride. The mixture became a colorless transparent solution after allowing to stand at room temperature for 5 hours. After adding 20 ml. of water, the reaction mixture was allowed to stand at room temperature for 2 hours. An oily material was obtained after removal of the solvent, and dissolved in 50 ml. of water to adjust to pH 5.0 with dilute sodium hydroxide. The solution was placed and passed on a column (1.5 x 20 cm.) of Amberlite CG-50 (H form). Development of the chromatogram with water afforded fractions positive to Lemieux reaction. A colorless powder (620 mg.) was obtained after removal of the solvent. The powder was dissolved in methanol (15 ml.) to remove insoluble material. To the filtrate was added ether until no precipitate was produced. This procedure was repeated twice, affording a colorless material, m.p. 143°-150°C., [ ]D20 +113° (c=l.l, H20) which was identified to be 2V,N -diacetyl derivative of 4. The yield was 313 mg. (0.79 mmole). Anal. Calcd. for C16H2809N2 C, 48.97 H, 7.19 O, 36.70 N, 7.14. Found C, 48.93 H, 7.46 O, 37.10 N, 6.92. 

Further development of the chromatogram with 0.1N ammonia afforded fractions positive to ninhydrin test. From the fractions, 249 mg. of a colorless material was obtained. It was dissolved in 19 ml. of water and the solution was adjusted to pH 4.0 with dilute hydrochloric acid. A colorless material, after condensing under a reduced pressure and lyophilization, was recrystallized from aqueous methanol with a small amount... 

Just prior to analysis the contents of the tube were evaporated to dryness. The residue was then re-dissolved in 100 pi of methanol and then diluted with 100 pi of 0.5 % acetic acid in water. 100 pi of the resulting mixture solution was then placed cm the column and the chromatogram obtained is shown in figure 9. It is seen that an excellent separation was obtained and again the retention and selectivity was dominated by dispersive interactions with the reversed phase. 

The sulfanes are soluble in carbon disulfide, benzene, tetrachloromethane, and dry diethylether (decreasingly so in that order) while alcohols and aqueous systems initiate rapid decomposition. For this reason a report on the chromatographic separation of the sulfanes H2S by reversed-phase HPLC using methanol as an eluent [35] was shown to be in error The peaks observed in the chromatogram have to be assigned to bismethoxy oligosulfanes... 

Induced fluorescence (X, > 430 nm, cut off filter) by thermal treatment of the chromatogram, the fluorescence increased by a factor of 2.5 by dipping in a solution of Triton X-100 — chloroform (1+4). Working range 2-50 ng substance per chromatogram zone. Prewashing the layers with methanol-ammonia solution (25%) (50+ 50) increased the precision. 

Note The individual components of the reagent can also be applied separately one after the other [12, IS], e.g. the chromatogram is first immersed in an 8% methanolic 4-(dimethylamino)-benzaldehyde solution and then, after intermediate drying, sprayed v th 25% sulfuric acid [12]. 4-(Dimethylamino)-benzaldehyde can be replaced in the reagent with 4-(dimethylamino)-ciimamaldehyde [1]. 

Analysis calculated for C1SH36N2O4S C, 57.41 H, 9.63 N, 7.43 S, 8.51. Found C, 57.60 H, 9.66 N, 7.37 S, 8.25. Thin-layer chromatograms (Note 10) run by the submitters showed a single spot for the product in each of three following solvent systems (solvents, volume ratio of solvents in the same order) chloroform-methanol-acetic acid, 85 10 5, Rf 0.60 1-butanol-acetic acid-water, 4 1 1, Rf 0.58 l-butanol acetic acid-pyridine-water, 15 3 10 12, Rf 0.71. 

---