Hydrolysate standard

Fig. 4-20. Separation of a hydrolysate standard on a totally sulfonated cation exchanger BTC 2710 (Biotronik, Maintal, Germany). — Column temperature four-step temperature program from 48 °C to 70 °C eluent 5 sodium citrate/borate buffers flow rate 0.3 mL/min for all other chromatographic conditions and elution order see Fig. 4-19.

Fig. 4-22. Separation of a hydrolysate standard on a latex anion exchanger AminoPac PA-1. -Eluent (A) 0.028 mol/L NaOH + 0.008 mol/L Na2B407, (B) 0.07 mol/L NaOH + 0.02 mol/L Na2B407, (C) 0.16 mol/L NaOAc, (D) 0.32 mol/L NaOAc, regenerent 0.56 mol/L NaOH + 0.64 mol/L H3B03 flow rate 1 mL/min detection see Fig. 4-19 injection 1 nmol of each of the various amino acids.

Figure 5.28 Separation of a hydrolysate standard on a totally sulfonated cation exchanger. Column LCA K06 temperature program 68-80 °C from 0-1 min, 80 °C from 1 -6 min, 80-66 °C from 6-9 min, 66 °C from 9-22 min, 66-74 °C from 22-47 min, 74 °C from 47-63 min, 74-64 °C from 63-68 min, and 64 °C until 76 min eluent buffer A-4/Na, A-5/...

Figure 5.29 Separation of a hydrolysate standard on a totally sulfonated cation exchanger. Column 1154110T temperature program 46°C from 0-4 min, 46-70 °C from 4-9 min, 70 °C from 9-32 min, 70-46 °C from 32-33 min eluent sodium citrate buffers gradient linear between pH 3.15,4.25, and 6.40 flow rate 0.60mL/min ...

The corresponding AminoPac PA-1 latexed anion exchanger, introduced at the same time as the AminoPac PC-1, has been replaced meanwhile by a more modern product, the AminoPac PA-10 (see Section 3.11). The baseline-resolved separation of 17 amino acids contained in a hydrolysate standard requires about 40 minutes, which is significantly longer than with a latex cation exchanger. 

The choice of type of derivative should be based on whether the chloride or anhydride is aliphatic or aromatic, because this factoi largely determines the reactivity. Aliphatic acid chlorides are best converted into their anilides, as in 4 above aromatic acid chloride may be similarly converted into their anilides, or they may be converted into their amides by shaking with an excess of ammonia (p, 120). (M.ps., pp. 544-545.) Aliphatic acid anhydrides should be converted into their crystalline anilides, but aromatic acid anhydrides arc best hydrolysed to the acid, which can then be converted into one of the standard derivatives (p. 349). 

The excess of unchanged acetic anhydride is then hydrolysed by the addition of water, and the total free acetic acid estimated by titration with standard NaOH solution. Simultaneously a control experiment is performed identical with the above except that the alcohol is omitted. The difference in the volumes of NaOH solution required in the two experiments is equivalent to the difference in the amount of acetic add formed, i.e., to the acetic acid used in the actual acetylation. If the molecular weight of the alcohol is known, the number of hydroxyl groups can then be calculated. 

C.HsNH, + CHjCO-O-COCHa = C.R NHCOCHs -h CHjCOOH then hydrolysed with water and the total free acetic acid estimated by titration with standard NaOH solution, the result being compared with that obtained in a control or blank experiment. 

In the analysis of oils and fats, where the quantity of fatty acid is the chief object of the deteiminalion, it is customary ttj hydrolyse the substance with a standard solution of aLoholic potash in place of aqueous potash, and to estimate the excess... 

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate (Eigure 23.7), like all phosphate ester hydrolyses, is a thermodynamically favorable (exergonic) reaction under standard-state conditions (AG° = —16.7 kj/mol). Under physiological conditions in the liver, the reaction is also exergonic (AG = —8.6 kJ/mol). Fructose-1,6-bisphosphatase is an allosterically regulated enzyme. Citrate stimulates bisphosphatase activity, hut fructose-2,6-bisphosphate is a potent allosteric inhibitor. / MP also inhibits the bisphosphatase the inhibition by / MP is enhanced by fructose-2,6-bisphosphate. 

As relatively few standard compounds are available from commercial or other sources, identification of flavonol glycosides has to be achieved by alternative means, for example UV-, H- and C-NMR spectroscopy. Therefore hydrolysing all glycosides to aglycones followed by HPLC determination offers a practical method for the quantitative determination of flavonoids in tea (Hertog et al, 1993a Wang and Helliwell, 2001). 

Characterization of various types of damage to DNA by oxygen-derived species can be achieved by the technique of gas chromatography-mass spectrometry (GC-MS), which may be applied to DNA itself or to DNA-protein complexes such as chromatin (Dizdaroglu, 1991). For GC-MS, the DNA or chromatin is hydrolysed (usually by heating with formic acid) and the products are converted to volatile derivatives, which are separated by gas chromatography and conclusively identified by the structural evidence provided by a mass spectrometer. Stable isotope-labelled bases may be used as internal standards... 

Berger [340] has examined the use of pSFC in polymer/additive analysis. As many polymer additives are moderately polar and nonvolatile SFC is an appropriate separation technique at temperatures well below those at which additives decompose [300,341,342], SFC is also a method of choice for additives which hydrolyse easily. Consequently, Raynor et al. [343] and others [284,344] consider that SFC (especially in combination with SFE) is the method of choice for analysing polymer additives as a relatively fast and efficient sample preparation method. Characterisation of product mixtures of nonpolar to moderately polar components encompassing a wide range of molecular masses can be accomplished by cSFC-FID. Unknown polymer additives may be identified quite adequately by means of cSFC-FID by comparison with retention times of standards [343], However, identification by this method tends to be time-consuming and requires that all the candidate compounds are on hand. SFC-FID of some low-to-medium polarity additives on reversed-phase packed columns... 

A GC analysis of amino acids requires a derivatisation step to increase the volatility of the amino acids. Generally, norleucine and/or norvaline are the internal standards added to the hydrolysate to check the derivatisation yield. According to the experimental method applied, the limits of detection (LOD) vary in the range 10 100 pg for each amino acid. Regarding the chromatographic columns, as most of the derivatives are esters barely polar compounds the most commonly used are fused-silica capillary columns with a low... 

The peptide/polypeptide product is usually hydrolysed by incubation with 6 mol l-1 HC1 at elevated temperatures (110 °C), under vacuum, for extended periods (12-24 h). The constituent amino acids are separated from each other by ion-exchange chromatography and identified by comparison with standard amino acid preparations. Reaction with ninhydrin allows subsequent quantification of each amino acid present. 

The analysis time for protein hydrolysates is 85 min using standard columns. For extra high resolution a high-resolution lithium cation exchange column is recommended which achieves baseline separation of virtually all 40 amino acids (Fig. 1.3). 

When magnesium sulphate was omitted from distilled water samples of phosphorus compounds, recovery was variable. Table 12.11 shows yields of a series of standards with and without the magnesium sulphate addition and with and without the final hydrolysis. The magnesium sulphate is used as an acidic solution (after addition to the seawater sample, the pH was about 3) to minimize silicate leaching from the glassware during evaporation. The acid and heating are necessary to hydrolyse any condensed phosphates in the final mixture. 

Hydrolysis and analysis The purified and dried polymer (0.1 g) and 1 ml of aqueous HC1 (0.1 mol l 1), together with a measured amount of acetone (as internal GLC standard), was heated for 1 h at 100 °C in a sealed tube to hydrolyse the polymer. The solution was then neutralised and the ethanol content was determined by GLC by means of a calibration graph. As polymers of 4 and 5 are strongly hydrophobic and are thus hydrolysed only very slowly in water, they were hydrolysed in aqueous dioxane. 

In the standard procedure the neutralised polymer is purified and hydrolysed and the hydrolysate is examined for ethanol. The absence of ethanol indicates that no tert.-oxonium ions containing a polymeric moiety and no polymeric oxycarbenium ions could have been present in the reaction mixture. 

The reactions were carried out in each case with a 0-1 per cent protein solution in phosphate buffer (pH 6 8), to which the radioactive phosphorofluoridate was added as a concentrated solution in dry ethanol. At the end of the reaction time, the product was dialysed for 20 hr. against running water, and precipitated at 0° by addition of two volumes of acetone. The precipitate was spun off and washed at —5° with ethanol and ether, and dried in air or over sulphuric acid. Samples of 25-50 mg. of dry powder were used for radioactivity determinations, and compared with a standard prepared by hydrolysing a weighed amount (ca. 1 mg.) of the phosphorofluoridate in n sodium hydroxide, neutralizing and drying. 

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