Wakopak^® Series for Amino Acid Analysis

Wakopak^® Wakosil^®-PTC

This column analyzes amino acid composition by quantifying the more quantitative PTC- amino acids obtained by the PITC labeling method. Gradient analysis is performed using two types of dedicated eluents (PTC-Amino Acids Mobile Phase), A and B. This method does not require a dedicated instrument, because it is a linear gradient rather than a complex gradient elution method as before. Therefore, it can be analyzed on most gradient HPLC systems.

Features

• Linear gradient elution with a dedicated eluent
• Can be analyzed using a general-purpose HPLC system
• High sensitivity: analysis up to 1 pmol possible
• Small interlot difference and high reproducibility

Examples of simultaneous analysis of 17 types of PTC-amino acids

• 

• Column	: Wakopak®Wakosil-PTC (4.0 mm x 200 mm)
  Eluent	: A) PTC-Amino Acids Mobile Phase A : B) PTC-Amino Acids Mobile Phase B 　linear gradient 　0 → 15 min. B 0 → 70%
  Flow Rate	: 1.0 mL/min. at 40 ℃
  Detection	: UV 254 nm 0.32AUS
  Sample	: PTC-amino Acids soln. (250 pmol)

Itsuo Inoue, Masami Fukumoto, Hitoshi Kamimori, Yoshitsugu Sakata, Shozo Shoji, Yukio Kubota: Biochemistry, 61, 1012 (1989).

PTC-amino acid derivatization method (method recommended by our company)

Derivatization procedure

1. 1. Transfer 10 μL (25 nmol) each of the sample (approximately 10 pmol to 500 nmol) and the Amino Acids Mixture Standard Solution, Type H (25 nmol) to an Eppendorf tube.
2. Dry under reduced pressure.
3. Add 20 μL of a mixture of ethanol, purified water, and triethylamine (TEA) (2/2/1) and stir.
4. Dry under reduced pressure.
5. Add 20 μL of a mixture of ethanol, purified water, TEA, and PITC (7/1/1/1) and stir.
6. Allow to react at room temperature for 20 minutes.
7. Dry under reduced pressure.
8. Store at -20℃ until analysis.
9. Dissolve in PTC-Amino Acids Mobile Phase A and proceed with analysis.
   * If the amino acid standard is dissolved in 1 mL and 10 μL of this solution is injected, the concentration is 250 pmol. Dissolve the sample in an appropriate quantity of PTC-Amino Acids Mobile Phase A and proceed with analysis.

• Derivatization reaction

  

• • When labeling samples, standard solutions must also be labeled at the same time, and quantitative calculation of amino acids in labeled samples must be performed using the analytical results of labeled standard solutions.
  • Labeled samples can be used for approximately 3 months when stored frozen (-20°C) in a dry state. The solution in PTC-Amino Acids Mobile Phase A can be used for several days when stored frozen (-20°C).

Wakopak^® Wakosil PTH-II φ4.6 mm x 250 mm (S-PSQ)

This column is for primary structural analysis of proteins and peptides. PTH-amino acids generated by Edman degradation can be analyzed with high reproducibility and sensitivity under isocratic conditions using a Wakopak^® Wakosil PTH-II φ4.6 mm × 250 mm (S-PSQ) and an eluate dedicated to PTH-amino acids (PTH-amino Acids Mobile Phase). It can also be applied to amino acids analysis in foods, drugs, media, and liquids by pretreatment appropriate for the sample.

Features

• Able to analyze 20 types of (PTH-derivatized) amino acids rapidly (within 25 minutes)
• Easy to operate (isocratic elution)
• Highly sensitive and reproducible analysis is possible

Examples of simultaneous analysis of 20 types of PTH- amino acids

Itsuo Inoue, Masami Fukumoto, Hitoshi Kamimori, Yoshitsugu Sakata, Naoki Morita, Takeshi Okumura: Biochemistry, 60, 876 (1988).

Wakopak^® Wakosil PTH-GR (S-PSQ) (for Gradient Elution)

This is a dedicated column used in the PPSQ series (gradient system), a fully automated primary protein structure analyzer (Shimadzu Corporation).
In the PPSQ-50, a fully automated primary protein structure analyzer (Shimadzu Corporation), the analysis is performed using an HPLC column following Edman degradation. The sample volume to be injected into the analytical column is 50 μL.
The chromatogram for the gradient elution of a PTH-amino acid mixture standard (2 pmol) is shown in Fig 1 (a), and the chromatogram for the isocratic elution is shown in Fig 1 (b). Overall, gradient elution enables the detection of peaks that are about 3 to 5 times higher than those of isocratic elution, allowing the separation of highly hydrophobic amino acids.

Wakopak^® Ultra APDS TAG^®

Wakopak^® Ultra APDS TAG^® is an UHPLC column optimal for analyzing derivatized amino acids witch are derivatized by Amino Acid Analysis Reagent (for LC/MS)(APDSTAG^®).

Analysis example

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  Analysis Conditions

  Column	Wakopak® Ultra APDS TAG®
  Flow rate	0.3 mL/min. at 40 ℃
  Detection	MS/MS, ESI positive, MRM
  Eluent A	APDSTAG Wako Eluent
  Eluent B	60% Acetonitrile

  
  

  Gradient

  Time (min)	0	0.05	1.7	1.71	4.95	5.95	5.96	6.7	6.71	12
  B(%)	6	8	8	12	30	60	95	95	6	6

• No.	Amino acid	Abbreviation	Q1	Q3
  1	2-Aminoethanol	EtOHNH2	182.1	121.0
  2	Glycine	Gly	196.1	121.0
  3	Sarcosine	Sar	210.0	121.0
  4	Alanine	Ala	210.1	121.0
  5	4-Aminobutyric acid	GABA	224.1	121.0
  6	3-Aminoisobutyric acid	β-AiBA	224.0	121.0
  7	2-Aminobutyric acid	α-ABA	224.0	121.0
  8	Serine	Ser	226.1	121.0
  9	Proline	Pro	236.1	121.0
  10	Valine	Val	238.1	121.0
  11	Threonine	Thr	240.1	121.0
  12	Taurine	Tau	246.1	121.0
  13	Hydroxyproline	Hypro	252.1	121.0
  14	Isoleucine	Ile	252.1	121.0
  15	Leucine	Leu	252.1	121.0
  16	Asparagine	Asn	253.1	121.0
  17	Aspartic acid	Asp	254.1	121.0
  18	Glutamine	Gln	267.1	121.0
  19	Glutamic acid	Glu	268.1	121.0
  20	Methionine	Met	270.1	121.0
  21	Histidine	His	276.2	156.0
  22	α-Aminoadipic acid	α-AAA	282.1	121.0
  23	Phenylalanine	Phe	286.2	121.0
  24	1-Methyl-L-histidine	1MeHis	290.2	170.0
  25	3-Methyl-L-histidine	3MeHis	290.2	170.0
  26	Arginine	Arg	295.2	175.1
  27	Citrulline	Cit	296.3	121.0
  28	Tyrosine	Tyr	302.2	121.0
  29	Tryptophan	Trp	325.2	121.0
  30	Carnosine	Car	347.2	227.1
  31	Anserine	Ans	361.3	241.1
  32	Ornithine	Orn	373.5	121.0
  33	Lysine	Lys	387.5	147.0
  34	5-Hydroxylysine	HyLys	403.5	163.0
  35	Cystathionine	Cysthi	463.2	223.1
  36	Cystine	Cys2	481.2	121.0