Hongcheng Wang, Bo Xu
Ngwa R&D Center
Okwu mmalite
Dị ka ndị ikwu polarities nke guzo n'akụkụ na mobile nkeji, mmiri chromatography nwere ike ikewa n'ime nkịtị frasi chromatography (NPC) na reverse chromatography (RPC).Maka RPC, polarity nke usoro mkpanaaka siri ike karịa nke oge akwụrụ.RPC abụrụla nke a na-ejikarị eme ihe na ụdị nkewa chromatography mmiri n'ihi oke arụmọrụ ya, mkpebi dị mma na usoro njide doro anya.Ya mere RPC dabara adaba maka nkewa na ịdị ọcha nke ogige polar dị iche iche ma ọ bụ ndị na-abụghị polar, gụnyere alkaloids, carbohydrates, fatty acids, steroid, nucleic acid, amino acids, peptides, proteins, wdg Na RPC, usoro nkwụsị nke a na-ejikarị eme ihe bụ. silica gel matrix nke jikọtara ya na otu dị iche iche na-arụ ọrụ, gụnyere C18, C8, C4, phenyl, cyano, amino, wdg N'ime ndị a na-arụ ọrụ na-ejikọta ọnụ, nke a na-ejikarị eme ihe bụ C18.A na-eme atụmatụ na ihe karịrị 80% nke RPC na-eji usoro nke C18 ugbu a.Ya mere kọlụm chromatography C18 aghọwo kọlụm ga-enwerịrị ogidi zuru ụwa ọnụ maka ụlọ nyocha ọ bụla.
Ọ bụ ezie na a pụrụ iji kọlụm C18 mee ihe n'ọtụtụ dịgasị iche iche nke ngwa, Otú ọ dị, maka ụfọdụ ihe atụ nke dị nnọọ polar ma ọ bụ nke ukwuu hydrophilic, ogidi C18 mgbe niile nwere ike inwe nsogbu mgbe ejiri ya mee ka ihe atụ dị ọcha.Na RPC, a na-enye iwu ihe mgbaze elution a na-ejikarị eme ihe dị ka polarity ha si dị: mmiri
Ọgụgụ 1. Ihe osise schematic nke usoro nke jikọtara n'elu silica gel na kọlụm C18 mgbe niile (aka ekpe) na C18AQ kọlụm (n'aka nri).
Iji dozie nsogbu ndị a kpọtụrụ aha n'elu, ndị na-emepụta ihe nkwakọba ihe chromatographic emeela nkwalite teknụzụ.Otu n'ime mmezi ndị a na-eme mgbanwe ụfọdụ n'elu elu nke silica matrix, dị ka ntinye nke ìgwè cyano hydrophilic (dị ka e gosiri na akụkụ aka nri nke Figure 1), iji mee ka elu nke silica gel dịkwuo hydrophilic.Ya mere, agbụ C18 dị n'elu silica nwere ike ịgbatị n'ụzọ zuru ezu n'okpuru ọnọdụ mmiri dị ukwuu ma nwee ike izere ọdịda hydrophobic.A na-akpọ kọlụm C18 ndị a gbanwetụrụ aqueous C18, ya bụ kọlụm C18AQ, nke emebere maka ọnọdụ elution nke ukwuu ma nwee ike ịnabata 100% sistemu mmiri.A na-etinye kọlụm C18AQ n'ọtụtụ ebe na nkewa na ịdị ọcha nke ogige pola siri ike, gụnyere organic acid, peptides, nucleosides na vitamin soluble mmiri.
Desalting bụ otu n'ime ahụkarị ngwa nke C18AQ ogidi na flash ọcha maka samples, nke na-ewepụ nnu ma ọ bụ ihe nchekwa ihe na sample ihe mgbaze ka ikwado ngwa nke sample na-esote ọmụmụ.Na post a, a na-eji FCF na-acha anụnụ anụnụ na-acha anụnụ anụnụ nwere polarity siri ike dị ka ihe nlele ma mee ka ọ dị ọcha na kọlụm C18AQ.Ihe mgbaze ihe nlere anya bụ ihe mgbaze organic si na nchekwa nchekwa dochie ihe mgbaze, si otú a na-eme ka evaporation rotary na-esote yana ịchekwa ihe mgbaze na oge ọrụ.Ọzọkwa, a na-emeziwanye ịdị ọcha nke ihe nlele ahụ site n'iwepụ ụfọdụ adịghị ọcha n'ime ihe nlele ahụ.
Ngalaba nnwale
Ọgụgụ 2. Usoro kemịkalụ nke ihe atụ.
Ejiri FCF na-acha anụnụ anụnụ dị ka ihe nlele na post a.Ịdị ọcha nke ihe nlele ahụ bụ 86% na usoro kemịkalụ nke ihe nlele ahụ gosipụtara na Figure 2. Iji kwadebe ihe ngwọta ihe atụ, 300 mg powdery crude siri ike nke Brilliant Blue FCF gbazere na 1 M NaH2PO4 ngwọta ngwọta ma maa jijiji nke ọma ịghọ. ngwọta doro anya kpamkpam.A na-agbanyezi ihe ngwọta nlele ahụ n'ime kọlụm flash site na injector.Edepụtara nhazi nnwale nke nchacha ọkụ na tebụl 1.
| Akụrụngwa | Igwe SepaBean™2 | |||
| Katrij | 12 g SepaFlash C18 RP flash katrij (silica sppherical, 20 - 45 μm, 100 Å, Nọmba iwu: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP flash cartridge (silica sppherical, 20 - 45 μm, 100 Å, Nọmba ịtụ: SW-5222-012-SP(AQ))) | ||
| Ogologo ogologo | 254 nm | |||
| Usoro mkpanaka | Ihe mgbaze A: Mmiri Ihe mgbaze B: methanol | |||
| Ọnụego mgbaba | 30 ml / min | |||
| Nbudata ihe atụ | 300 mg (FCF na-acha anụnụ anụnụ na ịdị ọcha nke 86%) | |||
| Gradient | Oge (CV) | Ihe mgbaze B (%) | Oge (CV) | Ihe mgbaze B (%) |
| 0 | 10 | 0 | 0 | |
| 10 | 10 | 10 | 0 | |
| 10.1 | 100 | 10.1 | 100 | |
| 17.5 | 100 | 17.5 | 100 | |
| 17.6 | 10 | 17.6 | 0 | |
| 22.6 | 10 | 22.6 | 0 | |
Nsonazụ na mkparịta ụka
A na-eji SepaFlash C18AQ RP flash katrij mee ihe maka ịsacha sachaa na sachaa.A na-eji nzọụkwụ gradient mee ihe nke a na-eji mmiri dị ọcha dị ka usoro mkpanaaka na mmalite elution ma na-agba ọsọ maka mpịakọta kọlụm 10 (CV).Dịka egosiri na eserese 3, mgbe ị na-eji mmiri dị ọcha dị ka usoro mkpanaka, a na-edobe ihe nlele ahụ kpamkpam na katrij flash.Na-esote, a na-abawanye methanol na usoro mkpanaka ozugbo na 100% ma debe gradient maka 7.5 CV.E wepụrụ ihe nlele ahụ site na 11.5 ruo 13.5 CV.N'ime ụmụ irighiri ihe ndị a chịkọtara, edobere ihe ngwọta nlele site na ngwọta nchekwa NaH2PO4 gaa na methanol.N'iji ya tụnyere ngwọta mmiri dị ukwuu, methanol dị mfe iwepụ site na evaporation rotary na nzọụkwụ na-esote, nke na-eme ka nyocha na-esote.
Ọgụgụ 3. chromatogram flash nke ihe nlele na katrij C18AQ.
Iji tụnyere omume njide nke katrij C18AQ na katrij C18 oge niile maka ihe atụ nke polarity siri ike, a na-eme nnwale ntụnyere yiri ya.A na-eji SepaFlash C18 RP flash cartridge mee ihe na chromatogram flash maka ihe nlele ahụ gosipụtara na eserese 4. Maka cartridges C18 mgbe niile, usoro nhazi mmiri kachasị elu bụ ihe dịka 90%.Ya mere etinyere gradient mmalite na 10% methanol na mmiri 90%.Dị ka egosiri na eserese 4, 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, ọrụ nke sample desalting ma ọ bụ ọcha enweghị ike mechaa.
Ọgụgụ 4. chromatogram flash nke ihe nlele na katrij C18 mgbe niile.
N'iji ya tụnyere linear gradient, iji nzọụkwụ gradient nwere uru ndị a:
1. Ihe mgbaze na-eji oge na-agba ọsọ maka nchacha ọcha na-ebelata.
2. Ngwaahịa a na-achọsi ike na-apụta na ọnụ ọgụgụ dị nkọ, nke na-ebelata ụda nke ụmụ irighiri ihe anakọtara ma si otú a na-eme ka ikuku rotary na-esote yana ịchekwa oge.
3. Ngwaahịa a na-anakọta dị na methanol nke dị mfe ikpochapụ, si otú a na-ebelata oge ihicha.
Na mmechi, maka ịsacha ihe nlele nke siri ike ma ọ bụ hydrophilic nke ukwuu, SepaFlash C18AQ RP flash cartridges na-ejikọta ya na sistemu chromatography nke na-akwadebe ngwa ngwa SepaBean ™ Machine nwere ike inye ngwọta ngwa ngwa na nke ọma.
Banyere katrij flash nke SepaFlash Bonded Series C18 RP
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.
logy (dị ka e gosiri na Tebụl 2).
Oge nzipu: Ọgọst-27-2018