Quantitative Determination of Residual Organic Solventｓ in　Peptides/Proteins

Organic solvent	LOQ	LOD
Dimethylformamide	145	43
Dimethylsulfide	48	14
Dimethylsulfoxid	45	13
Dioxane	100	70
Ethandithiole	343	103
Ethanol	58	18
Ethylacetate	45	14
2-Ethylbutanol	135	40
Heptane	33	10
HFIP	898	269
n-Hexane	61	18
Isohexane(I)	44	13
Isohexane(II)	44	13
Isohexane(III)	44	13
Isopropanol	42	12
Isopropylacetate	43	13
Methanol	44	13
Methylethylketone	59	18
Methyliodid	157	47
n-Methylmorpholine	82	25
n-Methylpyrrolidone	81	24

Organic solvent	LOQ	LOD
Pentane	103	31
Phenol	62	19
Piperidine	371	111
n-Propanol	62	19
Pyridine	61	18
t-Butanol	39	12
T-Butyl-Methylether	45	13
Tert-Butylnitrite	121	36
Tetrahydrofurane	57	17
Thioanisole	84	25
Toluene	28	8
Triethylamine	129	39
Triethylsilane	25	7
Triisopropylsilanol	42	13
Triisopropylsilane	65	20
Xylene(I)	28	8
Xylene(II)	28	8
Xylene(III)	28	8
DMPU	101	30
Sulfolane	79	24

Analysis:

Two samples are prepared:

• About 1 mg of sample is dissolved in 50 μl solvent free DMSO containing about 3000ppm Dioxane. The sample is shaken vigorously for a minute using a Vortex shaker.
• The reference solution contains 200 ppm to 5000 ppm of the solvents to be determined in 50 μL of solvent-free DMSO containing about 3000 ppm of dioxane.
• 50 μL of solvent-free DMSO containing about 3000 ppm of dioxane (internal standard solution).

The first sample is injected to calculate the content of the solvents using the internal standard method, the second to determine the retention times and/or response

factors, the third to verify that the solvents are not present in the internal standard solution.

Methods:

1. With the code 229 we determine the following solvents:

Methanol, ethanol, diethylether, acetone, 2-propanol, t-butanol, dichloromethane, acetonitrile, methyl-t-butyl ether, n-hexane, diisopropylether, methyl-ethylketone, ethylacetate, 2-butanol, chloroform, tetrahydrofuran, cyclohexane, isopropylacetate, heptane, n-butanol, toluene, pyridine, butylacetate, dimethylformamide, thioanisol, n-methyl-pyrrolidone, i-hexane, triethylamine, 2-ethylbutanol, xylene, triisopropylsilane, phenole, diisopropylamine (DIPEA), diisopropylamine (DIPA)

1. With the code 231 we analyze in addition to the above mentioned solvents: dioxane, anisole, dimethylacetamide (DMAA), diisopropylcarbodiimide (DIC), diimethylsulfoxide (DMSO) or in high sensitivity triethylamine, piperidine, triisopropylsilane or phenol. For determination of highly reactive components like DIC or formaldehyde spiking experiments are necessary to confirm the LOQ.

What we need: 5 mg of the sample