Amino acids standard,

Fig. 7-9. Separation of amino acids after derivatization 5 with OPA and mercaptoethanol. Column Superspher 100 RP-18 (4 pm) LiChroCART 250-4, mobile phase 50 mM sodium acetate buffer pH 7.0/methanol, flowrate 1.0 ml min temperature 40 °C detection fluorescence, excitation 340 nm/emission 445 nm. Sample amino acid standard sample (Merck KGaA Application note W219180).

Chromatographic procedures applied to the identification of proteinaceous paint binders tend to be rather detailed consisting of multiple analytical steps ranging from solvent extractions, chromatography clean up, hydrolysis, derivatisation reactions, and measurement to data analysis. Knowledge of the error introduced at each step is necessary to minimise cumulative uncertainty. Reliable results are consequently obtained when laboratory and field blanks are carefully characterised. Additionally, due to the small amounts of analyte and the high sensitivity of the analysis, the instrument itself must be routinely calibrated with amino acid standards along with measurements of certified reference proteins. All of these factors must be taken into account because many times there is only one chance to take the measurement. 

Figure 9.1 GC MS chromatograms acquired in the SIM mode of a laboratory blank (a) and an amino acid standard solution with concentrations at the quantitation limit (b). i.s.l, Hexadecane internal standard i.s.2, norleucine internal standard...

Figure 9.1a reporting GC-MS results taken on laboratory blank in a GC-MS procedure [87] shows negligible amino acid contamination. Indeed, quantitating a protein as the sum of 14 amino acids, a value of about 200 ng is the minimum required threshold to positively consider the protein identification. Figure 9. lb reports the chromatogram of an amino acid standard solution acquired in the SIM mode and corresponds to the quantitation limit that is a content of about 700 ng in the sample at a confidence level of 95%. This seems amenable when working at the trace level. 

HPLC analysis of hydrolyzed sound (bottom) and carious (middle) dentin of a tooth from series 11, and an amino acid standard (top). 

I. Amino acid standard (Pierce 20088) this is diluted tenfold with water prior to use. 

Reduced ninhydrin in solution tends to be unstable, therefore all kinds of precautions have to be taken, such as storage in the cold, in the dark, with a reducing agent added, and exclusion of oxygen. The condition of the ninhydrin solution has to be checked at regular intervals by running an aqueous amino acid standard mixture (Sigma) and adjustment of the response factors. 

Whatman 3MM paper, 20 X 20 cm Amino acid standard solutions, 1% in water Chromatography chambers... 

Create separate calibration curves for leucine and proline by plotting A570 or A440, respectively, versus equivalent amino acid standard concentration. 

Fig. 4 Elution position of S-3-aminopropylcysteine (AP-C) in four different amino acid analysis systems. Purified 5-3-aminopropylcysteine was spiked into amino acid standards and subjected to amino acid analysis. Standards run on (A) an AminoQuant (OPA) system, (B) a Waters PICO-TAG (PITC) chemistry system, (C) a Varian Amino Tag (FMOC) chemistry system, Y = tyrosine and FMOC-Cl, (D) a Beckman System 6300 (nin-hydrin) amino acid analyzer. (From Ref. 90. Copyright 1994 Academic Press, Inc.)...

Dabsyl Chloride (4-dimethyl-aminoazobenzene-4-sulfonyl chloride) Aabs = 420 nm. Derivatives are very stable (days) and can be formed from both primary and secondary amino acids. Detection is by absorption only. Detection limits are in the high picomole range. Reaction time is typically around 10 minutes at 70°C. Completeness of reaction can be adversely affected by the presence of high levels of various salts. Because reaction efficiency is highly matrix dependent and variable for different amino acids, standard amino acid solution should be derivatized under similar conditions/matrix for accurate calibration. Commercial systems available uti-... 

Fig. 11 Separation of amino acid standards derivatized with PITC. Eluent A sodium acetate/triethy-larnine buffer (pH 6.4) eluent B 60/40 acetonitrile/water. Pico-Tag column. Peak identification 1 Asp, 2 Glu, 3 Ser, 4 Gly, 5 His, 6 Arg, 7 Thr, 8 Ala, 9 Pro, 10 ammonia, 11 Tyr, 12 Val, 13 Met, 14 Cys, 15 lie, 16 Leu, 17 Phe, 18 Lys. (From Ref. 184. Copyright 1984 Elsevier Science.)...

TV-nitroso-fructosyl methionine, a nitroso sugar amino acid, standard. The latter gave two peaks, probably corresponding to its E and Z isomers, and each exhibited the same UV features as those of the standard when analyzed by a photodiode array detector (Fig. 6). 

Fig. 10.18. The separation of eighteen-amino-acids samples by pCEC. Column, PC-C18, 3 pm, 130 mm x 75 pm i.d. mobile phase (A), 50 mM sodium acetate-1% N,N-dimethyl-formamide, pH 6.4 (B), 50% acetonitrile in water linear gradient, 60-5% A in 6 min and kept at 5% 3,000V positive voltage across the column and 1,000 psi pressure were added on column during the separation flow-rate, 50 pl/min 20°C detection, 360 nm 20 nl injection. (A), 2 pg/ml of derived eighteen-amino-acids sample solution (B), 2 pg/ml of derived eighteen-amino-acids standard solution. Reproduced with permission from Ru et al. [197],...

Amino Acid Standard H (Sigma catalog AA-S-18) HPLC unit... 

Carefully spot 5 pi of the DNP-amino acid standards (provided by the instructor) along the origin of the plate. To prevent diffusion of the sample, it is a good idea to spot 1 pi of the sample, allow it to dry, spot another -pl aliquot, allow it to dry, and so forth. 

Using the same technique as described in step 6, carefully spot a 5-/xl aliquot of your sample along the same origin as the DNP-amino acid standards. If you did not recover a sufficient amount of the sample in the last ether extraction, you may also want to spot a 1 ()-pl sample next to this. The sample spot should be yellow enough in color that you will be able to identify it once the plate is developed. 

From the Rf value of your unknown sample compared to the Rf values of the DNP-amino acid standards, what is the identity of the N-terminal amino acid in your unknown dipeptide Discuss any uncertainty that may be involved in your identification, as well as how these uncertainties may be eliminated through modifications in the experiment. 

Spot 3 pi of each of the amino acid standards along the origin. As with the thin-layer chromatography plate, spot each sample 1 /u.1 at a time. Indicate the identity of each sample spot below the origin using a soft lead pencil. 

Calculate the Rf values of the amino acids in your unknown sample as well those of the amino acid standards, using the same procedure and equation as that used on Day 2. 

Transfer 10 pi of your acid-hydrolyzed dipeptide sample (colorless) to a microcentrifuge tube and add 20 pi of distilled water. In addition, pipette 0.2 5 ml of Amino Acid Standard H into a separate microcentrifuge tube. Perform steps 2 to 6 on both of these samples. 

Add 20 pX of wash reagent (ethanobtriethyl-amine water [2 2 1]), mix, and dry the sample down in the Speed-Vac under the high heat setting. Repeat this wash procedure one more time. The amino acids standards are supplied in a solution of 0.1 N HC1. The washes are performed to remove all traces of acid, which may interfere with the coupling reaction. 

Inject a 100-jul sample of the 20 PTC-amino acid standards and apply the same mobile-phase gradient. The sequence of the PTC-amino acids eluting from the column developed with this gradient is shown in Table 6-1. 

Measure the retention times (Rt) of the unknown PTC-amino acids and compare these to those of the PTC-amino acid standards. The retention time (Rt) can be calculated as follows ... 

DNP-amino acid standards (for thin-layer chromatography)—Dissolve 1 mg of each standard in 1.5 ml of... 

Lithium citrate buffers (Li-A, Li-B, and Li-C) and a lithium cation exchange column (20 X 4.6 cm) were purchased from Beckman. The amino acid standard was prepared from the Beckman standard with the addition of Hyl/flZZo-Hyl (from Sigma). Optimization of the method with respect to temperature and buffer change times was required to adequately resolve Hyl from ammonia. Hyl is partially converted to allo-Hyl during hydrolysis. The peak areas for Hyl and allo-Hyl were combined for standards and samples. 

---