Preparation of cryogel columns for depletion of hemoglobin from human blood

Figures & data

Figure 1. Effect of initial Ni(II) concentration on Ni(II) loading amount and optic photo of Ni(II) attached and plain PHEMA cryogel. IDA content: 8.27 mmol/g, flow rate: 1 mL/min, pH 7.0 HEPES, t: 2 h, T: 20 °C. m_{dry cryogel}: 0.41 g.

Figure 2. (a) SEM photographs of PHEMA cryogels with different magnifications; (a-1) × 500, (a-2) × 1000; (b). The FTIR spectra of PHEMA (top side), PHEMA–IDA (middle side), and PHEMA–IDA–Ni(II) (bottom side) cryogels.

Table 1. (a) Physicochemical properties of the PHEMA cryogel; (b) Coagulation times of human plasma (reported in second)*; (c) Langmuir and Freundlich adsorption constants and correlation coefficients for PHEMA–IDA–Ni(II) cryogel.

(a)
IDA attachment			8.27 mmol/g
Water uptake ratio			93%
Specific surface area			24.7 m^2/g
Pore size diameter			10–200 pm
Porosity			74.1% for PHEMA
				63.5% for PHEMA–IDA
Back pressure			0.26 MPa
(b)
	APTT	PT	CT
Control Plasma	32,0	16,5	19.0
PHEMA	28.3	15.2	17.4
PHEMA-IDA-Ni(ll)	27.8	15.0	16.8
(c)
	Experimental	Langmuir constants			Freundlich constants
		Qex (mg/g)	Qmax (mg/g)	b (g/mg)	R^2	KF	n	R^2
PHEMA–IDA–Ni(ll)	4.25	4.44	17.31	0.99	11.9	2.59	0.91

Figure 3. (a) Effect of pH on Hb adsorption amount. 0.1 M phosphate buffer (pH 7.4), flow rate: 0.5 mL/min, C_Hb: 0.05 mg/mL, m_{dry cryogel}: 0.41 g. T: 20 °C; (b) Effect of flow rate on Hb adsorption onto cryogels. 0.1 M phosphate buffer (pH 7.4), T: 20 °C, flow rate: 0.5 mL/min, C_Hb: 0.05 mg/mL, m_{dry cryogel}: 0.41 g; (c) Effect of ionic strength on Hb adsorption amount; 0.1 M phosphate buffer (pH 7.4), flow rate:0.5 mL/min, 0.05 mg/mL, m_{dry cryogel}: 0.41 g, t: 2 h, T: 20 °C; (d) Adsorption–desorption cycle showing the reusability potential of a column. 0.1 M phosphate buffer (pH 7.4), flow rate: 0.5 mL/min, C_Hb: 0.05 mg/mL, m_{dry cryogel}: 0.41 g, t: 2 h, T: 20 °C.

Figure 4. Adsorption behavior of Hb onto PHEMA–IDA–Ni(II) cryogel and modeling by Langmuir and Freundlich adsorption isotherms. Phosphate buffer (pH 7.4, 0, 1 M), flow rate: 0.5 mL/min, m_{dry cryogel}: 0.41 g, t: 2 h, T: 20 °C.

Table 2. Chromatographic separation data from human red blood cell hemolysate.

	tR (min)	N	k′	a	Rs
Hb	11.07	170.58	6.53	–	–
Other proteins	4.38	14.11	1.98	3.29	1.41

Figure 5. SDS-PAGE visualization for Hb depletion from human hemolysate. 5–12% SDS-PAGE, Lane 1: Hb standard, Lane 2: wide-range Sigma marker, Lane 3: crude sample of hemolysate, Lane 4: treated hemolysate with PHEMA–IDA–Ni(II) cryogel, Lane 5: Hb elution from the cryogel column.

Figure 6. FPLC separation of Hb from human hemolysate on a column packed with PHEMA–IDA–Ni(II) cryogel: Flow rate: 1.0 mL/min; detection was performed at 280 nm.