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ANALISIS ASAM AMNIO DAN PROTEIN. Abdul Rohman Fakultas Farmasi UGM. Bahan Pembelajaran Analisis asam amino dan Protein. Pendahuluan Analisis Asam amino Titrimetri Gasometri Spektrofotometri Kromatografi. Asam amino.

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analisis asam amnio dan protein

ANALISIS ASAM AMNIO DAN PROTEIN

Abdul Rohman

FakultasFarmasi UGM

bahan pembelajaran analisis asam amino dan protein
BahanPembelajaranAnalisisasam amino dan Protein
  • Pendahuluan
  • AnalisisAsam amino
    • Titrimetri
    • Gasometri
    • Spektrofotometri
    • Kromatografi
asam amino
Asam amino
  • AA merupakan monomer penyusun protein. Kebanyakan protein disusunoleh 20 asam amino
  • Sumberutama diet AA padamanusiaberasaldari protein, yang secaraenzimatisakandidigestiuntukmembebaskanAA-nya
  • AA dapatberadadalambentukbebasatauterikatdalampeptidaatauprotein menentukanmetodeanalisisdanpenyiapansampelnya
slide5

What is amino acid?

What is amino acid?

Amino Acid: aminated carboxylic acid (R-COOH)

R group

slide6

Examples

-amino

acetic acid

-amino propanoic acid

,-2 amino caproic acid

classification of amino acid
Classification of Amino Acid

1. By the location of Amino-group : / / -AA

2. By its acidity: neutral/acidic/ basic AA

ratio of Amino-group to carboxylic group

3. By whether containing phenyl group

aromatic / non aromatic AA

4. By its occurrence in protein

Protein / non protein AA

5. By polarity of R group :

polar / apolar side chain AA

6. By its nutrient value to human:

EssentialAA and non-essential AA

gasometri
Gasometri

Gugusalfa amino primer dariasam amino bereaksidenganasamnitritdanmenghasilkan gas nitrogen (N2).

Asamnitritinidibuatdenganmereaksikannatriumnitritdenganasamasetat.

Gas nitrogen yang terjadidimurnikandenganmengalirkannyapadakaliumpermanganat, untukselanjutnyadikumpulkandandiukurvolumenya.

spektrofotomeri uv
Spektrofotomeri UV

Aromatic AAs absorb light in the near ultraviolet (230-300nm).

phenylalanine

tyrosine

Note: This UV absorption property of protein is solely determined by the content of these 3 aromatic AAs. However, far ultraviolet (190nm)absorption of protein stems from the peptide bond.

tryptophan

spektroskopi visibel
Spektroskopivisibel

Ninhidrin

Metode yang dikembangkan

ninhidryn
Ninhidryn

FRIEDMAN, M. J. Agric. Food Chem. 2004, 52, 385−406

detektor
Detektor
  • Detektor UV-Vis merupakandetektor yang paling umumdigunakanuntukanalisisbahanmakananmenggunakan KCKT.
  • AA dapatdideteksisecaralangsungdengan UV karena AA mampumenyerapdipanjanggelombangantara 190 – 210. Meskipundemikian, karenakebanyakanpelarutdanjugakomponenmatrikssampel yang lain jugamenyerapdidaerahini, maka AA harusdiderivatisasiterlebihdahulusebelumdianalisisdengan KCKT menggunakandetektorspektrofotometer UV-Vis.
  • Derivatisasi AA dapatdilakukansebelumpemisahandikolomkromatografi (pre-column derivatization) ataudilakukansetelah AA keluardarikolom (post-column derivatization).
beberapa agen penderivat yang digunakan
Beberapaagenpenderivat yang digunakan

Dideteksidipanjanggelombang 570 nm

dansil klorida
Dansilklorida

Dansil-AA dideteksidenganfluoresensi (λeks 360 nm; λemisi = 470 nm) dandengan UV (λmaks 250 nm)

Source: Snyder et al., 1997

dabsil klorida
Dabsilklorida

Turunandabsilmenunjukkanserapanmaksimaldi 420 nm (daerahtampak), sertasangatstabil, danbatasdeteksinyaberadadi level pmol. Turunaninitetapstabilsetelahpenyimpananselama 4 minggudisuhuruang.

fenilisotiosianat
Fenilisotiosianat

Karenahasilderivatisasibersifattidakberfluoresensi, makateknikderivatisasidengan PITC dideteksidenganspektrofotometri UV-Vis, yang umumnyadilakukandi 254 nm (λmaks = 269 nm).

Meskipunderivatisasiinitidaksepekadibandingkandenganteknikderivatisasi yang menghasilkanderivat yang dideteksidenganfluoresen, akantetapiproduk PTC-AA bersifatsangatmenyerap UV, sehingga AA dengan level pmoldapatdideteksi.

Batas sensitifitasagenpenderivatiniadalah 50 pmolpadarasiosinyal/derau (S/N) 2,5

orto ftalaldehid
orto-ftalaldehid

Derivat yang dihasilkanantara AA dengan OPA dapatdideteksidengan UV padapanjanggelombang 330 nm, akantetapiuntukmenghasilkansensitifitas yang tinggi, makadigunakandetektorfluoresendenganλeks = 330 nm dan λ em = 430 nm.

slide34

Kromatogramstandar AA (atas) dan AA dalamsampelmadu (bawah). Deteksi UV = 280 nm). Konsentrasistandar AA (mg/kg) diberikandalamtanda kurung.1 = histidin (6,4); 2 = arginin (15,4); 3= asparagin (20,8); 4 = glutamin (41,8); 5 = serin (9,9); 6 = asamaspartat (25,3); 7 = asamglutamat (12,7); 8 = treonin (8,0); 9 = glisin (5,1); 10 = β-alanin (3,5); 11 = asam γ-aminobutirat (2,4); 12 = α-alanin (7,0); 13 = prolin (948,3); 14 = tirosin (15,7); 15 = metionin (3,5); 16 = valin (4,7); 17 = cistein (15,1); 18 = triptofan (7,2); 19 = ornitin (2,11); 20 = fenilalanin (93,2); 21 = isoleusin (14,5); 22 = leusin (15,5); 23 = lisin (7,8)

  • Source: Rebane, R. and Herodes, K. 2010. AnalyticaChimicaActa 672: 79–84
gas chromatography
Gas Chromatography

Principle:

Non-volatile AA

Volatile AA-derivate

automatic aa analyzer
Automatic AA analyzer

From pH2.2 to pH 6.4