Hongcheng Wang, Bo Xu
Cibiyar R&D Application
Gabatarwa
Dangane da ƙayyadaddun ƙayyadaddun lokaci na lokacin tsayawa da lokacin wayar hannu, ana iya raba chromatography na ruwa zuwa chromatography na al'ada (NPC) da jujjuyawar lokaci chromatography (RPC).Ga RPC, polarity na lokacin wayar hannu ya fi ƙarfin na lokaci na tsaye.RPC ya zama mafi yawan amfani da shi a cikin yanayin rabuwa na chromatography na ruwa saboda babban ingancinsa, ƙuduri mai kyau da ingantaccen tsarin riƙewa.Saboda haka RPC ya dace da rabuwa da tsarkakewa na daban-daban polar ko wadanda ba iyakacin duniya mahadi, ciki har da alkaloids, carbohydrates, m acid, steroids, nucleic acid, amino acid, peptides, sunadaran, da dai sauransu A cikin RPC, da aka fi amfani a tsaye lokaci ne. silica gel matrix wanda aka haɗa tare da ƙungiyoyi masu aiki daban-daban, ciki har da C18, C8, C4, phenyl, cyano, amino, da sauransu.An kiyasta cewa fiye da 80% na RPC yanzu suna amfani da lokacin haɗin C18.Saboda haka ginshiƙin chromatography na C18 ya zama dole ne ya sami shafi na duniya ga kowane ɗakin gwaje-gwaje.
Kodayake ana iya amfani da shafi na C18 a cikin aikace-aikace masu yawa, duk da haka, ga wasu samfurori wanda ke da iyakacin iyaka ko kuma hydrophilic, ginshiƙan C18 na yau da kullum na iya samun matsala yayin amfani da su don tsarkake irin waɗannan samfurori.A cikin RPC, ana iya yin oda da abubuwan da aka saba amfani da su bisa ga polarity: ruwa
Hoto 1. Tsarin tsari na matakan da aka haɗa a kan saman silica gel a cikin C18 na yau da kullum (hagu) da C18AQ shafi (dama).
Don magance matsalolin da aka ambata a sama, masana'antun tattara kayan chromatographic sun yi haɓaka fasaha.Ɗaya daga cikin waɗannan ingantawa yana yin wasu gyare-gyare a saman matrix silica, irin su gabatarwar ƙungiyoyin cyano na hydrophilic (kamar yadda aka nuna a cikin ɓangaren dama na Hoto 1), don sa saman silica gel ya fi hydrophilic.Don haka za a iya tsawaita sarƙoƙin C18 a saman silica gabaɗaya a ƙarƙashin yanayin ruwa sosai kuma ana iya guje wa rushewar lokaci na hydrophobic.Wadannan ginshiƙan C18 da aka gyara ana kiran su ginshiƙan C18 mai ruwa, wato C18AQ ginshiƙai, waɗanda aka tsara don yanayin haɓakar ruwa sosai kuma suna iya jure wa tsarin ruwa na 100%.An yi amfani da ginshiƙan C18AQ a cikin rarrabuwa da tsarkakewa na magungunan polar masu ƙarfi, ciki har da acid Organic, peptides, nucleosides da bitamin masu narkewa da ruwa.
Desalting yana ɗaya daga cikin aikace-aikace na yau da kullun na ginshiƙan C18AQ a cikin walƙiya mai walƙiya don samfurori, wanda ke kawar da abubuwan gishiri ko abubuwan buffer a cikin samfurin ƙarfi don sauƙaƙe aikace-aikacen samfurin a cikin karatun na gaba.A cikin wannan sakon, an yi amfani da FCF mai haske mai haske tare da polarity mai ƙarfi azaman samfurin kuma an tsarkake shi akan ginshiƙin C18AQ.An maye gurbin samfurin kaushi da kaushi na halitta daga maganin buffer, don haka sauƙaƙe ƙawancen jujjuyawar mai zuwa tare da adana kaushi da lokacin aiki.Bugu da ƙari kuma, an inganta tsabtar samfurin ta hanyar cire wasu ƙazanta a cikin samfurin.
Sashen Gwaji
Hoto 2. Tsarin sinadaran samfurin.
An yi amfani da Brilliant Blue FCF azaman samfurin a cikin wannan sakon.Tsabtataccen samfurin samfurin shine 86% kuma an nuna tsarin sinadarai na samfurin a cikin Hoto 2. Don shirya samfurin samfurin, 300 MG powdery crude m na Brilliant Blue FCF an narkar da shi a cikin 1 M NaH2PO4 buffer bayani kuma ya girgiza sosai ya zama. cikakken bayani bayyananne.Sa'an nan kuma an yi allurar maganin a cikin ginshiƙin filasha ta hanyar injector.An jera saitin gwajin filasha a cikin Tebur 1.
| Kayan aiki | Injin SepaBean™2 | |||
| Harsashi | 12 g SepaFlash C18 RP filashi mai walƙiya (silica silica, 20 - 45 μm, 100 Å, Lambar oda: SW-5222-012-SP) | 12 g SepaFlash C18AQ RP harsashin walƙiya (silica mai siffar zobe, 20 - 45 μm, 100 Å, Lambar oda: SW-5222-012-SP(AQ))) | ||
| Tsawon tsayi | 254nm ku | |||
| Zaman wayar hannu | Magani A: Ruwa Mai narkewa B: methanol | |||
| Yawan kwarara | 30 ml/min | |||
| Samfurin lodi | 300 MG (Brilliant Blue FCF tare da tsarki na 86%) | |||
| Gradient | Lokaci (CV) | Mai narkewa B (%) | Lokaci (CV) | Mai narkewa 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 | |
Sakamako da Tattaunawa
An yi amfani da harsashin walƙiya na SepaFlash C18AQ RP don lalata samfurin da tsarkakewa.An yi amfani da matakin gradient wanda a ciki aka yi amfani da ruwa mai tsafta azaman lokacin wayar hannu a farkon elution kuma ana gudanar da kundin shafi 10 (CV).Kamar yadda aka nuna a hoto na 3, lokacin amfani da ruwa mai tsafta azaman lokacin wayar hannu, samfurin ya kasance gabaɗaya a kan harsashin filasha.Na gaba, methanol a cikin tsarin wayar hannu ya karu kai tsaye zuwa 100% kuma an kiyaye gradient don 7.5 CV.An cire samfurin daga 11.5 zuwa 13.5 CV.A cikin ɓangarorin da aka tattara, an maye gurbin samfurin samfurin daga maganin buffer NaH2PO4 zuwa methanol.Idan aka kwatanta da mafi yawan maganin ruwa, methanol ya kasance mafi sauƙi don cirewa ta hanyar ƙazantaccen juyawa a mataki na gaba, wanda ke sauƙaƙe bincike mai zuwa.
Hoto 3. Hasken chromatogram na samfurin akan harsashi na C18AQ.
Don kwatanta halayen riƙewa na harsashi C18AQ da harsashi na C18 na yau da kullun don samfuran polarity mai ƙarfi, an yi gwajin kwatancen layi ɗaya.An yi amfani da harsashin filasha na SepaFlash C18 RP kuma an nuna chromatogram mai walƙiya don samfurin a cikin Hoto na 4. Don harsashi na C18 na yau da kullun, mafi girman jure yanayin yanayin ruwa shine kusan 90%.Don haka an saita gradient na farawa a 10% methanol a cikin 90% ruwa.Kamar yadda aka nuna a cikin Hoto na 4, saboda rugujewar lokaci na hydrophobic na sarƙoƙi na C18 da ke haifar da babban rabo mai ruwa, da kyar aka riƙe samfurin a kan harsashi na C18 na yau da kullun kuma lokacin wayar hannu ya ɓace kai tsaye.A sakamakon haka, aikin samfurin desalting ko tsarkakewa ba za a iya kammala.
Hoto 4. chromatogram mai walƙiya na samfurin akan harsashin C18 na yau da kullun.
Idan aka kwatanta da layin layi, yin amfani da matakin gradient yana da fa'idodi masu zuwa:
1. Amfani da ƙarfi da lokacin gudu don samfurin tsarkakewa ya ragu.
2. Samfurin da aka yi niyya ya ƙare a cikin ƙaƙƙarfan kololuwa, wanda ke rage yawan ɓangarorin da aka tattara kuma don haka sauƙaƙe ƙawancen jujjuyawar mai zuwa tare da adana lokaci.
3. Samfurin da aka tattara yana cikin methanol wanda ke da sauƙin cirewa, saboda haka an rage lokacin bushewa.
A ƙarshe, don tsarkakewar samfurin wanda yake da ƙarfi polar ko mai tsananin ruwa, SepaFlash C18AQ RP filashin filashi yana haɗuwa tare da tsarin chromatography na shirye-shiryen SepaBean ™ Machine na iya ba da mafita mai sauri da inganci.
About the SepaFlash Bonded Series C18 RP flash cartridges
Akwai jeri na SepaFlash C18AQ RP flash cartridges tare da ƙayyadaddun bayanai daban-daban daga Fasahar Santai (kamar yadda aka nuna a Table 2).
| Lambar Abu | Girman Rukunin | Yawan kwarara (ml/min) | Max.Matsi (psi/bar) |
| SW-5222-004-SP(AQ) | 5.4g ku | 5-15 | 400/27.5 |
| SW-5222-012-SP(AQ) | 20 g | 10-25 | 400/27.5 |
| SW-5222-025-SP(AQ) | 33g ku | 10-25 | 400/27.5 |
| SW-5222-040-SP(AQ) | 48g ku | 15-30 | 400/27.5 |
| SW-5222-080-SP(AQ) | 105 g | 25-50 | 350/24.0 |
| SW-5222-120-SP(AQ) | 155 g ku | 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 |
Tebur 2. SepaFlash C18AQ RP filasha harsashi.
Abubuwan da aka shirya: Babban inganci mai siffar siffar C18 (AQ) - silica mai ɗaure, 20 - 45 μm, 100 Å.
logy (kamar yadda aka nuna a Table 2).
Lokacin aikawa: Agusta-27-2018