Test mixtures chromatogram

Figure 5. Effect of column length and flow rate on preparative HPLC separation. Gradient test mixture chromatograms using linear gradient elution from 10% to 90% aqueous methanol containing 0.1% TEA. UV absorbance monitored at 220 nm. (a) 20 X 250 mm column, 10 mL min flow rate, 30 min gradient time, (b) 20 x 100 mm column, 20 mL min flow rate, 10 min gradient time, (c) 20 x 100 mm column, 40 mL min flow rate, 5 min gradient time, (d) 20 x 50 mm column, 20 mL min flow rate, 5 min gradient time.

Having chosen the test mixture and mobile diase composition, the chromatogram is run, usually at a fairly fast chart speed to reduce errors associated with the measurement of peak widths, etc.. Figure 4.10. The parameters calculated from the chromatogram are the retention volume and capacity factor of each component, the plate count for the unretained peak and at least one of the retained peaks, the peak asymmetry factor for each component, and the separation factor for at least one pair of solutes. The pressure drop for the column at the optimum test flow rate should also be noted. This data is then used to determine two types of performance criteria. These are kinetic parameters, which indicate how well the column is physically packed, and thermodynamic parameters, which indicate whether the column packing material meets the manufacturer s specifications. Examples of such thermodynamic parameters are whether the percentage oi bonded... 

Figure 4.10 Typical routine column test chromatogram for a 30 cm X 4.6 mm I. D. column pacXed with an octadecylsiloxane bonded silica packing of lO micrometers particle diameter. The test mixture consisted of resorcinol (0.55 mg/ml), acetophenone (0.025 mg/ml), naphthalene (0.20 mg/ml) and anthracene (0.01 mg/ml) in acetonitrile, 10 microliters injected. The separation was performed isocratically at 23 C with acetonitrile-water (55 45) as the mobile phase at a flow rate of 1.5 ml/min. Detection was by UV at 254 nm (0.1 AUFS).

Figure 7.44 shows the 2D UV chromatogram (RPLC-UV/VIS (DAD)) for a five-compound test mixture of polymer additives [662]. Any spectral data collected during hyphenated chromatography-spectroscopy measurements can be readily transformed into 2D correlation spectra. 

Figure 7.44 2D UV chromatogram (254 nm) for a five-compound test mixture. After Louden et al. [662]. Reproduced from D. Louden et al., Anal. Bioanal. Chem., 373, 508-515 (2002), by permission of Springer-Verlag, Copyright (2002)...

When the column is ready to be used, the chromatogram of a suitable test mixture should be obtained. The plate number and retention times of the test solutes should be noted, and the peaks should have a satisfactory shape (minimal tailing). For measurement of the plate number, the recorder should be used at a high chart speed. Fig. 5.1b(i) and (ii) show test chromatograms for a C-18 column prepared by the above method, and Fig. 5.1c and 5.Id show the data that you should report with the chromatogram. The retention for an unretained peak is taken as the small baseline disturbance just before the first peak. 

A laboratory technician tests a liquid mixture with gas chromatography for the purpose of identifying the components. She injects the mixture and four peaks are displayed on the chromatogram. She then obtains four pure liquids from a stock room and injects them into the GC (same conditions) one at a time. The retention time of one of the pure liquids exactly matches one of the retention times on the mixture chromatogram. Do you think she now knows, with certainty, the identity of one of the components Explain. 

Fig. 3.130. HPLC chromatograms of the test mixture detected by DAD (270 nm, upper lane) by APCI-MS-TIC (middle) and by ESI-MS-TIC (lower lane). Peak identification l=benzene sulphonic acid sodium salt 2=2-naphtalene sulphonic acid sodium salt 3=2-anthraquinone sulphonic acid sodium salt 4 = sulphorhodamine D sodium salt 5=crocein orange G 6=eriochrome black T 7=2,6-anthraquinone disulphonic acid disodium salt 8 = 1,5-naphtalene disulphonic acid disodium salt 9 = azophloxine 10 = 1,2-benzene disulphonic acid dipotassium salt. Reprinted with permission from G. Socher et al. [178].

FIGURE 9.4 Chromatograms of a reversed-phase test mixture. Plot A is the chromatogram at 30°C and ImL/min, and plot B is the chromatogram at 100°C and 5mL/min. Solutes 1, uracil 2, p-nitroaniline 3, methyl benzoate 4, phenetole 5, toluene. 2.1% (w/w) poly-butadiene coated zirconia column, mobile phase 20% ACN, flow rate was l.OmL/min at 30°C and 5mL/min at 100°C. detection 254 nm. (Reprinted from Li, J. et al., Anal. Chem., 69, 3884, 1997. Copyright 1997, American Chemical Society. With permission.)... 

Dry the sheet again and keep on the vapor of concentrated HCl. Identify the red spots in comparison with a chromatogram of DABTH amino acid test mixture and determine migration relative to the bluish spots of the markers (schematic drawing of a 2 x 2-cm TLC plate is given in Fig. 3.1). 

LC columns are fairly durable unless they are used in some manner that is intrinsically destructive, as, for example, with highly acidic or basic eluents, or with continual injections of inadequately purified biological samples. It is wise to inject some test mixture into a column when new, and to retain the chromatogram. If questionable results are obtained later, the test mixture can be injected again under the specified conditions. The two chromatograms may be compared to establish whether the column is still useful. 

The use of the laser-light-scattering detector permits the record of excellent chromatograms in gradient elution. An analysis of the test mixture of standards is shown in Fig. 37B for comparison with the same analysis obtained in isocratic conditions. The analysis lasts about 30 min before the elution of BBB instead of 18 min in isocratic conditions, but the first part of the chromatogram is considerably improved, and a number of impurities in the standards can be resolved. 

Fig. 37 Chromatograms of a test mixture of homogeneous tryglyceride standards. Columns 250 X 4-mm ID LICHROSPHERERP18 (5 /rm) constant flow rate l.Oml/min. A, isocratic conditions solvent chloro-form/acetonitrile (49 51 v/v) (one column). B, gradient elution chloroform in acetonitrile. Starting conditions 30 70, program rate 1%/min for 25 min to 55 45 and then 3%/min for 15 min to 100 0 and hold at 100% chloroform for 8 min (two columns in series). Some impurities are contained in the standard compounds.

Figure 15.11 (a) Total ion chromatogram of a Grob test mixture obtained on an Rtx-1701... 

Figure 6.1 UV chromatograms of the test mixture of four / -hydroxybenzoic acid esters (1, methyl 2, ethyl 3, propyl 4, butyl) after the column and after the NMR flow cell at flow rates of (a) 1.0 and (b) 0.1 ml/min conditions column, LiChrospher RP select B, 125 x 4 mm id, 5 Jim, spectrometer, Bruker DRX 600 probe head, 4 mm z-gradient LC probe, active volume 120 a1 eluents, acetonitrile (A) and D2O (B) gradient, t = Omin A/B (40/60), t = 8 min A/B (70/30) at a flow rate of 1.0 ml/min and t = 80 min A/B (70/30) at a flow rate of 0.1 ml/min...

Purge line A with water and line B with MeOH. Dial-a-mix 70% MeOH and equilibrate the C18 column at l.OmL/min. When stable, inject 15pL of the seven-component test mixture and annotate the chromatogram s start. Run an isocratic chromatogram. 

Inject 15j L of the seven-component standards test mixture. Annotate and run an isocratic chromatogram. 

Put acetonitrile in the B reservoir. Purge the pump inlet line with acetonitrile. Dial-a-mix 60% acetonitrile/water. Reconnect the C18 column at O.lmL/min. Increase the flow to l.OmL/min and equilibrate the column. Inject 15 pL of the seven-component test mixture. Annotate and run the chromatogram. 

Detector output is recorded as a function of time, producing a chromatogram, which consists of a series of peaks on a time axis. Each peak represents a compound in the vaporized test mixture, although some peaks may overlap. The elution time is characteristic of the individual compounds (qualitative analysis), and the peak area is a function of the amount present (quantitative analysis). 

Fig. 2.3. Chromatogram of 2 jil aliquois of a test mixture on a 100 x 2.1 mm column of 3.5 jcm SymmetryShield RPI8. Mobile phase A was 0.191 v/v formic acid in water and mobile phase B was 0.19 v/v formic acid in acetonitrile-water (95 5 v/v). Gradicnl time was 5 min and the flow rate was 1.5 ml/min. The temperature was 60°C. giving a starting pressure of ca. 300 bar (4350 psi). Detection was at 215 nm. Peak identities pyridine, quinine, ben/yl alcohol, phenol, nortriptyline, acetophenone. 3-methyl-4-nitrobenzoic acid, methyl salicylaldehyde, 4-chlorocinnamic acid and octanophenone.

Harke and Drews used a 50 m long stainless steel capillary column, 0.5 mm I.D., coated with Ucon LB 550 X (polyethylene glycol) and K0H for the separation of tobacco alkaloids. A typical chromatogram of a test-mixture containing 3-pyridyl-n-propylketon, nicotine, nor-nicotine, myosmine, anabasine and nicotyrine is shown in Figure 5.1 (Tobacco alkaloids). 

Fig. 3.17 Chromatogram of a test mixture to assess hydrophobic properties, efficiency and shape selectivity of a RP material (eluent methanol-water, 75 25 v/v).

Figure 3.31 shows a typical chromatogram of the test mixture described in Tab. 3.16 on a dc = 50mm column packed with LiChrospher Si 60, 12 pm. 

For water treatment purposes, only small amounts of chlorine dioxide are necessary therefore, the reaction products chlorite and chlorate are only present at low concentrations. Their separation from the main components is performed with a separator column IonPac AS9. Fig. 8-7 shows the chromatogram of a test mixture developed by the American Environmental Protection Agency (EPA). This test mixture is adjusted to the matrix of such water, thus allowing to test the applied chromatographic method with regard to separation efficiency and sensitivity. 

Paper chromatography was another popular method of studying organic water constituents (Shapiro, 1957 Packham, 1964 Midwood and Felbeck, 1968) and also was of limited value in characterizing humic substances and colored organic acids, because the complex mixtures often produced broad diffuse bands and streaks. Some of the spot-tests and chromatograms of Shapiro (1957) demonstrated the presence of colorless acids and suggested the presence of phenols and enols. 

Figure 8. Chromatograms of testing mixture obtained on the columns A) filled with CPG (Sbet = 347 m /g D= 13.4 nm Vp = 1.16cm /g), B) packed with LiChrosorb Si-100 (Sbet = 318m /g D = 16.0 nm Vp = 1.32 cm /g). Column 250 x 4 mm. Mobile phase hexane at 1 ml/min. Peaks 1 = benzene 2 = naphthalene 3 = diphenyl 4 = anthracene 5 = nitrobenzene.

A specific test mixture may be prepared for working in a special area, e.g. an aflatoxin mixture for aflatoxin determination. (However, a test mixture for the determination of the plate number should not include any peptides or proteins. These compounds need to be eluted by gradient, see Figure 18.7, whereas the plate number is calculated from an isocratic chromatogram.) In all other cases, a test mixture that satisfies the following criteria is needed ... 

Figure 9.3 Separation of a test mixture on silica with eluents of different strength. Top Ze/ f-butylmethyl ether, ° = 0.48 middle hexane-fezt-butylmethyl ether (9 1), = 0.29 bottom hexane, = 0. The attenuation ofthe hexane chromatogram is only half as much as of the other two. Conditions column, 25cm x 3.2 mm i.d. stationary phase, LiChrosorb SI 60 5 im flow rate, 1 ml min UV detector, 254 nm. Peaks 1 =p-xylene 2 = nitrobenzene 3 = acetophenone 4 = 2,6-dinitrotoluene.

If the test sample is introduced simultaneously into two paralled columns connected to two detectors assembled in a differential circuit (or to two cells of one heat-conduction detector), and a reaction tube is connected in series with one of the columns, the chromatogram will show only the peaks of the components being removed [9]. This method is possible only when the test mixture is removed from the two columns simultaneously,... 

Fig. 5.5 depicts two chromatograms. The chromatogram on column A contains all of the components of the test mixture tert.-butanol (1), -butanol (2), -cymol (a), linalool... 

Fig. 5.5. Chromatograms of initial mixture (column A) and test mixture after separation and subtrac-...

Tosio et al. [70] suggested that in order to identify and determine acid components in mixtures with neutral compounds, a flow of carrier gas should be passed through a reactor (100 X 0.5 cm l.D.) packed with potassium hydroxide on a quartz powder (115 100) after the chromatographic separation of the initial mixture on an analytical column. Selective absorption of the acid components takes place in this reactor. By comparing the chromatogram obtained with the analytical column and that obtained with an alkaline reactor, it is possible to identify and determine the acid and neutral components of the test mixture. As an example, results were presented of the analysis of small amounts of... 

Fig. 3.9. Gas chromatograms (FID) of test mixtures on AR-glass capillary columns. (1) Grob test mixture on OV-21S. Initial temperature 70°C, programmed at 5°C/min. Peaks Cn = undecane ol = octanol P = 2.6-dimethylphenol s = 2-ethylhexanoic acid al = nonanal A = 2,6-dimelhylaniline am = dicyclohexylamine E,o, E, and E,j = C,q, C, and C,2-acid methyl esters. (2) Nitrophenol test on SE-52. Initial temperature 100°C, programmed at 7°C/min. Peak assignment o, m, p = oriho,meta,para-nilTophenol , 2,4, 2,6 = 2,4-dinitrophenol and 2,6-dinitrophenol. (3) Diamine test on SE-30. Initial temperature 60°C, programmed at 7°C/min. Peaks dh = 1,6-diaminohexane do = 1,8-diaminooctane. Reproduced from [lOS].

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