Chromatograms separations

FIGURE 1-2. A typical chromatogram. Separation of Mountain Dew soft drink sample peak 1 is caffeine and peak 2 is benzoic acid. The two unmarked peaks are unidentified components. (Reproduced from reference 1 with permission.)... 

Fig. 31.8 A multicomponent chromatogram. Separation of many compounds, some well resolved, e.g. peaks at 12-13 mins, and others that are not, e.g. peaks at 24-25 mins.

Fig. 23.—Radioautograph of paper chromatogram separation of mixture obtained by treating radioactive n-gliicose and inactive dextriii with R. marcrans amylase. Gi, Ga, Os, etc., represent n-glucose, maltose, maltotriose, etc. a, 0, y represent the positions to wliicdi the Sehardinger dextrins move. Note that the Schardinger dextrins them.selves do not become radioactive. After the chromatogram was sectioned, inactive a-dextrin was isolated from the band between G4 and Gs and identified by means of the iodine test.

Table 1 Relationship between sample dimensionality and chromatogram separation pattern...

Fig. 1.10. Amino acid chromatogram. Separation of a mixture of amino acids (10 nmol/amino acid) by an amino acid analyzer. Applied is a single ion exchange column Durrum DC-4A, 295 x 4 mm buffers Pi/P2/P3- 0.2 N Na-citrate pH 3.20/0.2 N Na-citrate pH 4.25/1.2 N Na-citrate and NaCl of pH 6.45. Temperatures T1/T2/T3 48/56/80 °C. Flow rate 25 ml/h absorbance reading after color development with ninhydrin at 570/440 nm ...

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. 

Direct property prediction is a standard technique in drug discovery. "Reverse property prediction can be exemplified with chromatography application databases that contain separations, including method details and assigned chemical structures for each chromatogram. Retrieving compounds present in the database that are similar to the query allows the retrieval of suitable separation conditions for use with the query (method selection). 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

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... 

Although aimed at the introductory class, this simple experiment provides a nice demonstration of the use of GG for a qualitative analysis. Students obtain chromatograms for several possible accelerants using headspace sampling and then analyze the headspace over a sealed sample of charred wood to determine the accelerant used in burning the wood. Separations are carried out using a wide-bore capillary column with a stationary phase of methyl 50% phenyl silicone and a flame ionization detector. 

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. 

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. 

Two gas chromatograms showing the effect of polarity of the stationary phase on the separation efficiency for three substances of increasing polarity toluene, pyridine, and benzaldehyde. (a) Separation on silicone SE-30, a nonpolar phase, and (b) separation on elastomer OV-351, a more polar phase. Note the greatly changed absolute and relative retention times the more polar pyridine and benzaldehyde are affected most by the move to a more polar stationary phase. 

Schematic diagram showing the injection of a mixture of four substances (A, B, C, D) onto a GC column, foliowed by their separation into individuai components, their detection, and the dispiay (gas chromatogram) of the separated materiais emerging at different times from the coiumn.

A single peak from an ordinary gas chromatogram (a) is revealed as two closely separated peaks by resolution enhancement (b). 

An example of a size-exclusion chromatogram is given in Figure 7 for both a bench-scale (23.5 mL column) separation and a large-scale (86,000 mL column) mn. The stationary phase is Sepharose CL-6B, a cross-linked agarose with a nominal molecular weight range of 5000-2 x 10 (see Fig. 6) (31). 

Fig. 7. Chromatograms of size-exclusion separation of IgM (mol wt = 800,000) from albumin (69,000) where A—D correspond to IgM aggregates, IgM, monomer units, and albumin, respectively, using (a) FPLC Superose 6 in a 1 x 30 — cm long column, and (b) Sepharose CL-6B in a 37-cm column.

Fig. 11. Simulated chromatograms of chiral separations obtained using nonchiroptical detection (a, c, e) and chiroptical detection (b, d, f) illustrating the...

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