Amino Acids standard analysis

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. 

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

Period 2 Part B—Work up the dansyl hydrolysate and spot on the TLC plate with standard dansyl amino acids. Part A. 1—Work up peptide hydrolysate and prepare FMOC derivatives of amino acids for analysis by HPLC or CE. Part A.2-If applicable, develop paper chromatogram in solvent system. 

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.)...

Amino acid standard (2.5 mM) for hydrolysate analysis (Beckman Ltd)... 

Quantitative Ion-Exchange Column Chrom. tographic Analysis OF AN Amino Acid Standard Solution... 

Filtrate (total amino acids) or supernatant (free amino acids) from above was diluted (20 1) with 0.2 N lithium citrate buffer, pH 2.8, prior to analysis using a Beckman 6380 amino acid analyzer (Beckman Coulter, Inc.) equipped with a 10 cm ion exchange column and lithium citrate buffer supplied by Beckman. Detection was via post column derivatization using ninhydrin. Calibration was via mixed external free amino acid standards. 

Figure 13.20 HPLC of amino acid derivatives detected by 254 nm UV absorption (a) 200 pmol of PTC-amino acid standard, including pbospboserine (PH-S), pbospbothreonine (PH-T), bydroxy-proline (OH-P), galactosamine (Gal), norleucine (NLE, 1 nmol internal standard), excess reagent (Re), and other amino acids designated by one letter codes listed in Fig. 13.19 and (b) Analysis of a human fingerprint, taken up from a watchglass using a mixture of water and ethanol. (Courtesy of National Gallery of Art and the Andrew W. Mellon Foundation.) (Cazes, used with permission.)...

After purification, polymers are typically characterized by a standard set of techniques that can include amino acid content analysis, mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immu-noblotting. These methods are intended to verify the identity of the polymer. Depending on the type of polymer being... 

As mentioned in Section III, separation techniques for amino acids designed originally for PC are generally applicable for TLC amino acid studies on cellulose. Solvent systems for the TLC of amino acids generally employ mixtures of alcohols, acids or bases, and water. TLC is advantageous compared to PC for amino acid analysis in that it is faster and provides more compact spots, leading to better sensitivity and resolution of compounds. Experiment 1 provides a simple introduction to the TLC analysis of amino acid standards on silica gel. Experiment 2 provides a similar experience with cellulose and amino acid standards. Experiment 3 uses a reversed-phase layer to separate amino acids. For TLC exper-... 

Figure 9 Microscale analysis of amino acid standards and 10 s microdialysate fractions (120 nL sample collected from live rat brain). Blank of saline (bottom trace) and an amino acid standard (top trace) were diluted to 2 pL and derivatized in a polypropylene microvial. Concentrations of the amino acids (nM) ASP (10), GLU (20), ASN (20), HIS (40), GLN (500), CIT (20), ARG (50), SER (100), O-phosphoelthanolamine (PEA, 20), THR (50), GLY (100), TYR (20), L-ALA (100), P-alanine (P-ALA, 25), TAU (50), and GABA (10). Inset is expanded portion of the chromatogram where GABA elutes. For all experiments, an injection volume of 250 nL was used. Separation and detection conditions are as described in Figure 8 except the mobile phase pH was 6.51.

Figure 11 Ultra-trace level analysis of an amino acid standard using the automated, capillary-scale amino acid analyzer. A 2 pL, low nanomolar amino acid standard (top trace) and blank (bottom trace) is derivatized and preconcentrated using the automated amino acid analyzer. The samples were contained in a polypropylene, 384-weU microtiter plate. The reagents were added, mixed, and siphoned for injection using the fused silica needle of the autosampler (Figure 14). The time axis is truncated to highlight the elution window of the amino acid derivatives. All separation and detection parameters are as described in Figure 9.

FIGURE 9 Amino acid analysis of a monoclonal antibody (mAb) using liquid-phase acid hydrolysis (6 M HCI), followed by AccQ-tag labeling and UHPLC separation. The top panel shows a chromatogram of amino acid standards. 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

The standard protocol for analysis of the amino acid composition of proteins is discussed in Section 5.1. Results of such analyses allow the researcher to anticipate which methods of polypeptide fragmentation might be useful for the protein. 

Because the amount of time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. Figure 26.3 shows the results of amino acid analysis of a standard equimolar mixture of 17 a-amino acids. Typically, amino acid analysis requires about 100 picomoles (2-3 /xg) of sample for a protein containing about 200 residues. 

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