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- Posts: 1
- Joined: Thu May 28, 2020 6:55 am
Dear all,
I have a problem with recovery in low level.
HPLC, LUNA C18 column. Eluents: A- 1 ml H3PO4 in 1L water, B - 1 ml H3PO4 in 1L MeOH. 223 nm for protein (low absorbance), 254 nm for IP.1 and EEDQ.
Sample consist of protein (M=907.96) - it is starting material for IP.1, IP.1 itself and EEDQ (also starting material). This sample is dissolved in DMF:ACN (1:9) and diluted. LINEARITY solution and ACCURACY solution prepared in the same manner, same concentration (protein, IP.1, EEDQ), same dilution procedure. Problem is that LINEARITY solution have protein area, which is twice smaller than in ACCURACY solution. And this trend in all linearity solutions. IP.1 and EEDQ areas are not changing. Why?
I have no carry-over, no precipitation, freshly prepared. Please help, if You can.
I have a problem with recovery in low level.
HPLC, LUNA C18 column. Eluents: A- 1 ml H3PO4 in 1L water, B - 1 ml H3PO4 in 1L MeOH. 223 nm for protein (low absorbance), 254 nm for IP.1 and EEDQ.
Sample consist of protein (M=907.96) - it is starting material for IP.1, IP.1 itself and EEDQ (also starting material). This sample is dissolved in DMF:ACN (1:9) and diluted. LINEARITY solution and ACCURACY solution prepared in the same manner, same concentration (protein, IP.1, EEDQ), same dilution procedure. Problem is that LINEARITY solution have protein area, which is twice smaller than in ACCURACY solution. And this trend in all linearity solutions. IP.1 and EEDQ areas are not changing. Why?
I have no carry-over, no precipitation, freshly prepared. Please help, if You can.
