Reversedphase High Performance Liquid Chromatography

The speed and sensitivity of reversed-phase high performance liquid chromatography (RP-HPLC) has led to significant developments in the analysis of proteins: RP-HPLC is now one of the most widely used techniques for the analysis of amino acids, since pre-column derivatization is possible with a selection of derivatizing agents and a variety of detection techniques can be employed. RP-HPLC lends itself well to conservation science, being particularly suitable for the analysis of the extremely small samples removed from works of art.

Phenylthiocarbamyl derivatives of amino acids in the hydrolysate of proteinaceous media samples have been separated on a C18 column using a ternary solvent system as the mobile phase (water-acetonit-rile-acetate buffer).

Following hydrolysis of proteinaceous material removed from a series of Italian 15th-century painted panels, Halpine used phenyl isothiocyanate (PITC) for the derivatization of the amino acids, which were then separated on a C18 column using a binary solvent system of acetonitrile and acetate buffer (Table 2). The addition of nor-leucine as an internal standard facilitated the quantification of the amino acid components in the proteins, which in turn resulted in the characterization of a number of animal glue and egg/glue media. However, PITC-amino acid derivatives degrade in solution, so must be stored at low temperature prior to analysis.

PITC derivatives have also been analysed by RP HPLC when attempting to identify media samples which had been removed from a variety of French and Italian stone and wooden sculptures, frescoes and statues. Proteinaceous material was extracted from the matrices with sodium hydroxide and the subsequent analysis indicated the presence of gelatine and egg proteins.

9-Fluorenylmethyl chloroformate (FMOC) is a useful derivatizing agent for amino acids since it favours mild, aqueous conditions, reacts with both primary and secondary amino acids and is stable at room

Table 2 Percentage amino acid composition

Percentage amino acid composition

Control samples Samples from panels

Percentage amino acid composition

Control samples Samples from panels

Amino acid

Control 1

Control 2

Control 3

Control 4

Sample 1 Sample 2 Sample 3

Sample4

Aspartic acid

1.0

9.1

5.6

7.9

5.5

*

10.2

5.2

Glutamic acid

2.3

11.1

9.3

13.0

5.9

6.4

9.8

4.1

Hydroxyproline

12.3

0.6

0.3

11.7

9.9

3.8

5.9

Serine

3.8

11.1

10.7

9.4

5.7

5.7

8.4

8.9

Glycine

27.7

10.9

7.8

5.2

26.9

24.7

15.8

18.6

Histamine

0.8

1.3

1.5

1.6

*

*

*

1.2

Arginine

5.7

5.3

5.9

4.0

4.1

*

4.3

3.0

Threonine

2.6

5.6

6.6

3.9

3.9

*

4.8

6.6

Alanine

11.1

8.6

10.2

8.8

11.8

11.2

10.0

12.8

Proline

16.9

5.4

5.9

4.0

11.6

12.4

6.7

10.3

Tyrosine

1.2

2.7

3.6

2.4

*

*

1.1

1.8

Valine

3.3

6.6

7.8

7.5

4.2

5.3

5.5

7.2

Methionine

1.3

1.7

2.0

4.8

*

6.7

1.3

0.3

Isoleucine

1.9

4.3

5.0

5.8

3.6

11.7

4.7

4.6

Leucine

3.9

8.5

10.0

9.0

1.5

6.0

7.2

7.1

Phenylalanine

2.0

3.3

3.7

5.9

3.7

*

3.3

2.4

Lysine

2.0

4.0

4.5

5.7

*

*

3.0

*

Too small to quantify; control 1, rabbit skin glue ground with blue pigment; control 2, egg yolk with red pigment; control 3, egg yolk with blue pigment; control 4, egg albumin; sample 1, light blue paint; sample 2, dark blue paint; sample 3, light brown paint; sample 4, green paint. All paint samples removed from Cosimo Tura's The Annunciation with Saint Francis and Saint Louis of Toulouse (c. 1475). Reproduced from Halpine (1992) with permission.

Too small to quantify; control 1, rabbit skin glue ground with blue pigment; control 2, egg yolk with red pigment; control 3, egg yolk with blue pigment; control 4, egg albumin; sample 1, light blue paint; sample 2, dark blue paint; sample 3, light brown paint; sample 4, green paint. All paint samples removed from Cosimo Tura's The Annunciation with Saint Francis and Saint Louis of Toulouse (c. 1475). Reproduced from Halpine (1992) with permission.

temperature for up to 2 weeks. The FMOC moiety is both highly fluorescent and a good UV chromophore, so absorption and emission detection techniques can be used. Detection methods are an important concern in conservation science in view of the very small samples available - FMOC is particularly useful since fluorescence is usually far more sensitive than UV absorption. Standard proteinaceous media and museum sample hydrolysates have been characterized as FMOC derivatives.

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