Rui Huang, Bo Xu
Ngwa R&D Center
Okwu mmalite
A peptide bụ ihe mejupụtara amino asịd, nke ọ bụla n'ime ha nwere ihe pụrụ iche nke anụ ahụ na kemịkal n'ihi ụdị dị iche iche na usoro nke ihe fọdụrụ amino acid mejupụtara usoro ya.Site na mmepe nke njikọ kemịkalụ siri ike, njikọ kemịkalụ nke peptides dị iche iche na-arụ ọrụ emeela nnukwu ọganihu.Otú ọ dị, n'ihi mgbagwoju anya nke peptide nwetara site na nhazi usoro siri ike, ngwaahịa ikpeazụ kwesịrị ịdị ọcha site na ụzọ nkewa pụrụ ịdabere na ya.Ụzọ nchacha a na-ejikarị maka peptides gụnyere ion mgbanwe chromatography (IEC) na reverse-phase high performance liquid chromatography (RP-HPLC), nke nwere adịghị ike nke obere sample loading ikike, ọnụ ahịa mgbasa ozi nkewa, mgbagwoju anya na ọnụ ahịa nkewa. wdg. Maka ncha ngwa ngwa nke obere peptides molecule (MW <1 kDa), Santai Teknụzụ bipụtara akwụkwọ anamachọihe gara nke ọma, nke ejiri SepaFlash RP C18 cartridge mee ihe maka nhicha ngwa ngwa nke thymopentin (TP-5) na enwetara ebumnuche ngwaahịa a chọrọ.
Ọgụgụ 1. 20 amino asịd nkịtị (nke ewepụtara na www.bachem.com).
Enwere ụdị amino acid 20 nke a na-ahụkarị na ngwakọta nke peptides.Enwere ike kewaa amino acid ndị a n'ime otu ndị a dịka polarity ha na ihe onwunwe acid-base: ndị na-abụghị polar (hydrophobic), polar (uncharged), acidic ma ọ bụ isi (dị ka egosiri na Figure 1).N'usoro peptide, ọ bụrụ na amino acid mebere usoro ahụ na-abụkarị ndị polar (dị ka akara na agba pink na eserese 1), dị ka Cysteine, Glutamine, Asparagine, Serine, Threonine, Tyrosine, wdg mgbe ahụ peptide a nwere ike inwe ike siri ike. polarity na-ukwuu soluble na mmiri.N'oge usoro nchacha maka ihe atụ peptide pola siri ike site na chromatography na-agbanwe agbanwe, ihe a na-akpọ ọdịda hydrophobic ga-eme (tụ aka na ndetu ngwa e bipụtara na mbụ nke Santai Teknụzụ: Hydrophobic Phase Collapse, AQ Reversed Phase Chromatography Columns and Applications Ha).E jiri ya tụnyere kọlụm C18 mgbe niile, kọlụm C18AQ emelitere kacha dabara adaba maka ịsacha ihe atụ polar siri ike ma ọ bụ hydrophilic.Na post a, ejiri peptide polar siri ike dị ka ihe nlele wee sachaa ya site na kọlụm C18AQ.N'ihi ya, ngwaahịa ezubere iche na-enweta ihe achọrọ ma nwee ike iji ya mee nchọpụta na mmepe ndị a.
| Ngwa | SepaBean™igwe 2 | |||
| Katrij | 12 g SepaFlash C18 RP flash katrij (silica sppherical, 20 - 45 μm, 100 Å, Nye iwu nunber: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash katrij (silica sppherical, 20 - 45 μm, 100 Å, Nọmba ịtụ: SW-5222-012-SP(AQ)) | ||
| Ogologo ogologo | 254 nm, 220 nm | 214 nm | ||
| Usoro mkpanaka | Ihe mgbaze A: Mmiri Ihe mgbaze B: Acetonitrile | |||
| Ọnụego mgbaba | 15 ml / min | 20 ml / min | ||
| Nbudata ihe atụ | 30 mg | |||
| Gradient | Oge (CV) | Ihe mgbaze B (%) | Oge (nkeji) | Ihe mgbaze B (%) |
| 0 | 0 | 0 | 4 | |
| 1.0 | 0 | 1.0 | 4 | |
| 10.0 | 6 | 7.5 | 18 | |
| 12.5 | 6 | 13.0 | 18 | |
| 16.5 | 10 | 14.0 | 22 | |
| 19.0 | 41 | 15.5 | 22 | |
| 21.0 | 41 | 18.0 | 38 | |
| / | / | 20.0 | 38 | |
| 22.0 | 87 | |||
| 29.0 | 87 | |||
Nsonazụ na mkparịta ụka
Iji tụnyere arụmọrụ nchacha maka ihe nlele peptide polar n'etiti kọlụm C18 mgbe niile na kọlụm C18AQ, anyị na-eji kọlụm C18 mgbe niile maka ịsacha ọkụ nke ihe nlele ahụ dị ka mmalite.Dị ka egosiri na eserese 2, n'ihi ọdịda hydrophobic nke agbụ C18 nke oke mmiri dị elu kpatara, a na-edobe ihe nlele ahụ na katrij C18 oge niile ma usoro mkpanaka wepụrụ ya ozugbo.N'ihi ya, ihe nlele ahụ ekewaghị nke ọma ma mee ka ọ dị ọcha.
Ọgụgụ 2. chromatogram flash nke ihe nlele na katrij C18 mgbe niile.
Ọzọ, anyị na-eji a C18AQ kọlụm maka flash ọcha nke sample.Dị ka egosiri na foto 3, a na-ejigide peptide nke ọma na kọlụm wee wepụ ya.Ekewapụrụ ngwaahịa ezubere iche na ihe adịghị ọcha dị na ihe nlele raw wee chịkọta ya.Mgbe lyophilization wee nyochaa ya site na HPLC, ngwaahịa a na-eme ka ọ dị ọcha nwere ịdị ọcha nke 98.2% na enwere ike iji ya mee ihe maka nyocha na mmepe ọzọ.
Ọgụgụ 3. chromatogram flash nke ihe nlele na katrij C18AQ.
Na mmechi, SepaFlash C18AQ RP flash katrij jikọtara ya na flash chromatography sistem SepaBean™igwe nwere ike na-enye ihe ngwọta ngwa ngwa na nke dị irè maka ịdị ọcha nke polar siri ike ma ọ bụ ihe atụ hydrophilic.
Enwere usoro nke SepaFlash C18AQ RP flash cartridges nwere nkọwa dị iche iche sitere na teknụzụ Santai (dị ka egosiri na Tebụl 2).
| Nọmba ihe | Nha kọlụm | Ọnụego mgbaba (mL/min) | Oke. Nrụgide (psi/bar) |
| SW-5222-004-SP(AQ) | 5,4 g | 5-15 | 400/27.5 |
| SW-5222-012-SP(AQ) | 20 g | 10-25 | 400/27.5 |
| SW-5222-025-SP(AQ) | 33 g | 10-25 | 400/27.5 |
| SW-5222-040-SP(AQ) | 48 g | 15-30 | 400/27.5 |
| SW-5222-080-SP(AQ) | 105 g | 25-50 | 350/24.0 |
| SW-5222-120-SP(AQ) | 155 g | 30-60 | 300/20.7 |
| SW-5222-220-SP(AQ) | 300 g | 40-80 | 300/20.7 |
| SW-5222-330-SP(AQ) | 420 g | 40-80 | 250/17.2 |
Tebụl 2. SepaFlash C18AQ RP flash katrij.Ihe nkwakọ ngwaahịa: C18 (AQ) na-arụ ọrụ nke ọma, silica jikọtara ọnụ, 20 - 45 μm, 100 Å.
Maka ozi ndị ọzọ gbasara nkọwa zuru ezu nke igwe SepaBean™, ma ọ bụ ozi ịtụ ihe na sepaFlash series flash cartridges, biko gaa na webụsaịtị anyị.
Oge nzipu: Ọktoba 12-2018