BSA 蛋白吸附 解析
Journal of Biotechnology79(2000)259–268
BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states
Willem Norde a,Carla E.Giacomelli a,*,1
a Laboratory of Physical Chemistry and Colloidal Science,Wageningen Uni6ersity,Dreijenplein6,
6703HB Wageningen,The Netherlands
Received9August1999;accepted24November1999
Abstract
The secondary structure and the thermostability of bovine serum albumin(BSA),before adsorption and after homomolecular displacement from silica and polystyrene particles,are studied by circular dichroism spectroscopy and differential scanning calorimetry.The structural perturbations induced by the hydrophilic silica surface are reversible, i.e.BSA completely regains the native structure and stability after being exchanged.On the other hand,the adsorption on,and subsequent desorption from,polystyrene particles causes irreversible changes in the stability and (secondary)structure of BSA.The exchanged proteins have a higher denaturation temperature and a lower enthalpy of denaturation than native BSA.The a-helix content is reduced while the b-turn fraction is increased in the exchanged molecules.Both effects are more pronounced when the protein is displaced from less crowded sorbent surfaces.The irreversible surface-induced conformational change may be related to some aggregation of BSA molecules after being exposed to a hydrophobic surface.?2000Published by Elsevier Science B.V.All rights reserved.
Keywords:Bovine serum albumin;Adsorption;Desorption;Homomolecular exchange;Thermostability;Secondary structure
https://www.wendangku.net/doc/612872917.html,/locate/jbiotec
1.Introduction
When an aqueous solution containing protein is exposed to another(solid,liquid or gas)phase,it leads in almost all cases to adsorption of the protein at the interface between the two phases (Norde and Lyklema,1991).The overall protein adsorption process comprises various steps or stages:transport of the protein from the bulk solution into the interfacial region,attachment of the protein at the sorbent surface and the relax-ation of the protein on the surface,i.e.optimisa-tion of the protein-surface interaction.Depending on the extent of relaxation the molecule may detach more or less readily(Ball et al.,1998). The adsorption of proteins on solid surfaces is often described as‘irreversible’since,after attach-ment is established,which has usually reached a ?nal value within an hour,subsequent replace-ment of the protein solution by pure solvent,as a rule,does not lead to any signi?cant desorption
*Corresponding author.
1On leave from the Departamento de Fisicoqu?′mica,Facul-
tad de Ciencias Qu?′micas,Universidad Nacional de Co′rdoba,
Co′rdoba,Argentina.
0168-1656/00/$-see front matter?2000Published by Elsevier Science B.V.All rights reserved. PII:S0168-1656(00)00242-X
W.Norde,C.E.Giacomelli/Journal of Biotechnology79(2000)259–268 260
on a time scale of hours or even days.However,it does not mean that protein molecules remain attached to the surface whatever the conditions of the solution in which the surface is immersed. With respect to reversibility of adsorption/desorp-tion of proteins,distinction should be made be-tween reversibility toward dilution of the solution (Norde and Anusiem,1992;Norde and Haynes, 1995),changes in pH and ionic strength(Galisteo and Norde,1995;Kondo and Fukuda,1998), addition of other types of surface-active sub-stances(Norde et al.,1986;Norde and Favier, 1992)and exchange against dissolved proteins (Brash et al.,1983;Ball et al.,1994;Huetz et al., 1995;Bentaleb et al.,1997).Although desorption upon dilution does not occur,protein molecules may be released from the surface by other surface active molecules added to the system by an ex-change mechanism in which the protein molecules are replaced from the surface in favour of adsorp-tion of other molecules.Perhaps,the most clear example of such an exchange process is the‘Vro-man effect’:the transient adsorption of proteins from blood plasma,in which,the more abundant smaller proteins are displaced by the less abun-dant larger proteins that have higher af?nities for the surface(Vroman and Adams,1986).The ex-change process between protein molecules in the adsorbed state and in solution can be hetero-molecular,like the Vroman effect,or homomolec-ular,as in systems where a large amount of one kind of protein is present in excessive amounts so that it is accommodated both in the adsorbed state and in solution.Then,protein adsorption is a dynamic process by which protein molecules are continually exchanged between the adsorbed and the dissolved states.It is well known that relax-ation at the sorbent surface involves more or less perturbation of the original,native protein struc-ture.The question arises whether the molecules returning from the surface into the solution regain their original structure.
The aim of this work is to investigate the secondary structure and the thermostability of bovine serum albumin(BSA)after homomolecu-lar exchange from two sorbent surfaces with dif-ferent hydrophobicities(silica and polystyrene particles).These systems are model-systems which are not directly related to a particular practical application.They were selected because the at-tachment and relaxation stages of BSA on these surfaces have been described(Elgersma et al., 1990;Kondo et al.,1991,1992;Kondo and Hi-gashitani,1992;Norde and Favier,1992)and the exchange of albumin between the adsorbed and dissolved state has been demonstrated by using labelling(radio and?uorometric)techniques (Brash and Samak,1978;Lok et al.,1983;Fraaije, 1987).However,whether the protein regains its native structure after homomolecular exchange has received much less attention.The protein structure was studied by differential scanning calorimetry(DSC),which provides the heat of thermal denaturation and the denaturation tem-perature,and by circular dichroism spectroscopy (CD),from which the secondary structure of the protein can be judged.These two techniques are complementary,as DSC provides information on a macroscopic scale and CD information on a molecular level.Thus,by comparing the struc-tural properties of the protein before adsorption with those after exchange,information is obtained on the reversibility of the homomolecular ex-change process.
2.Material and methods
2.1.Sorbent surfaces
PS?is a negatively charged polystyrene latex which was prepared as described elsewhere (Goodwin et al.,1974,1979).Silica particles are a commercial product(Aerosil OX-50,Degussa). The speci?c surface areas of the solids have been determined by the BET method;the electrokinetic potentials have been established by electrophore-sis.The physicochemical properties of the sorbent that are considered to in?uence protein adsorp-tion are summarised in Table1.
2.2.Protein and others chemicals
Bovine serum albumin,BSA,was purchased from Sigma(A-7030)and used as received.Table 1contains those properties of the protein which
W.Norde,C.E.Giacomelli/Journal of Biotechnology79(2000)259–268261
may be relevant to its adsorption behaviour. Buffer solutions were prepared by mixing appro-priate volumes of10mM Na2HPO4and10mM NaH2PO4solutions to give pH7.All other chem-icals used were of analytical grade.
2.3.Adsorption isotherms
The adsorption experiments were performed in 10ml polypropylene centrifuge tubes each con-taining the same amount of sorbent material. Phosphate buffer solution and protein stock solu-tion were added to the sorbent to give a series of samples of constant volume but varying protein concentration.Then,the tubes were rotated end-over-end overnight at room temperature.After incubation,the samples were centrifuged and the protein concentration in the supernatant was mea-sured using UV spectroscopy.The amount of protein adsorbed was calculated from material balance(depletion method).
The solution before exposing to the sorbent as well as the supernatant obtained after centrifuga-tion were analysed by DSC and CD to investigate changes in the structural properties of the proteins induced by having been in contact with the sor-bent surfaces.In order to follow the in?uence of the degree of coverage of the sorbent surface on the structural changes,different supernatant solu-tions along the isotherms were selected.
2.4.Differential scanning calorimetry(DSC) Calorimetry experiments were made using a Setaram Micro-DSC III calorimeter(Setaram, Caluire,France).A total of1–3mg of protein in solution,either before adsorption or after being exchanged from the sorbent surface,were placed into the1ml measurement cell while the1ml reference cell was?lled with the appropriate blank material(buffer or buffer which was in contact with the sorbent).Then,the cells were stabilised at20°C inside the calorimeter before heating up to90°C with a scanning rate of0.5°C min?1. Cooling and reheating of the samples showed that the denaturation process occurred irreversibly. The denaturation temperature and the enthalpy of denaturation were determined using the Setaram software(version1.3).The DSC data reported are the mean values of at least two measurements deviating less than10%for the enthalpies and less than1°C for the temperatures.
2.5.Circular dichroism(CD)
The CD spectra were recorded on a JASCO spectropolarimeter,model J-715(JASCO,Tokyo, Japan).A quartz cuvette of0.1cm path length was used.The spectra were scanned between190 and260nm with0.2nm resolution;16scans were accumulated with a scan rate of100nm min?1 and a time constant of0.125s.The spectral analysis was performed by?tting the measured spectra with reference spectra based on the CD curves of poly-L-lysine containing varying amounts of a-helix,b-sheet,b-turn and random coil conformations.The reference spectra were described by Green?eld and Fasman(1969)and Chang et al.(1978).Fitting of the measured spec-tra was performed by a non-linear regression pro-cedure,making use of the Gauss–Newton algorithm(de Jongh et al.,1994).The experimen-
Table1
Some physicochemical properties of the protein and the sor-bent surfaces used
Protein BSA
Molar mass67000
4.7
Isoelectric point
(pH units)
Heart shaped of 8nm of side Molecular dimensions
and 3nm of depth Conformational High degree of secondary characteristics structure(mainly a-helix);low
thermal stability
PS?Sorbent surface Silica
(supplied as colloidal
dispersion)
?O??SO3?Charged group
?48?69 Electrokinetic potential
(mV)
Contact angle of a82°
0–5°
H2O drop
8
Speci?c surface area42
(m2g?1)
Medium10mM phosphate buffer pH7
W .Norde ,C .E .Giacomelli /Journal of Biotechnology 79(2000)259–268
262Fig.1.Adsorption isotherms of BSA adsorbed on ( )silica and on ( )PS ?particles at pH 7(phosphate buffer 10mM)and room temperature.
terised by well-de?ned plateau values.The ad-sorption isotherms of BSA on both silica and latex are in good agreement with the results ob-tained under practically the same conditions by Elgersma et al.(1990),Norde and Favier (1992)and Kondo et al.(1992).
Figs.2and 3compare the DSC thermograms of BSA before adsorption and after being ex-changed from silica and PS ?particles,respec-tively.The data derived from the thermograms are collected in Table 2.As the BSA molecule comprises three different structural domains,three discernible transitions in the thermal denat-uration might be expected.However,the ther-mograms showed only one single peak.From this peak,the denaturation enthalpy,D D H ,and tem-perature,T D ,are evaluated as the peak area and the temperature at half-peak area,respectively.
Fig.2.DSC thermograms of BSA ( )before adsorption and ( )after being in contact with silica particles.The DSC thermograms obtained after BSA was exchanged from silica particles at different surface coverage coincide with the one displayed on the ?gure.
tal spectra were ?tted with 1-nm resolution in the 190–240nm range and no constraints were used in the procedure.The quality of the ?t was ex-pressed by the normalised root-mean-square (RMS)error as described by Brahms and Brahms (1980).A ?t was considered reliable when the RMS error was smaller than 10.
3.Results
The adsorption of BSA on silica and PS ?latex particles are presented in isotherms,as shown in Fig.1,where the adsorbed amount (G )is plotted as a function of the protein concentration in solution after adsorption (c BSA ).The adsorbed amount of BSA (in mass per unit sorbent surface area)at pH 7is invariant with the sorbent used.The initial slopes of the isotherms do not differ signi?cantly and adsorption saturation is charac-
W.Norde,C.E.Giacomelli/Journal of Biotechnology79(2000)259–268263
Fig.3.DSC thermograms of BSA( )before adsorption and after being exchanged from PS?particles at different surface coverage:( )G=1.1mg m?2;( )G=0.9mg m?2;(") G=0.8mg m?2.al.,1977;Svein et al.,1979;Yamasaki et al., 1990),namely a T D-value of57°C and a D D H of 11J g?1.After being exchanged,at any surface coverage,from silica particles the thermal stability of BSA is not changed.On the other hand,the DSC curves for BSA exchanged from latex parti-cles deviate from the one of the protein before adsorption:T D increases and D D H decreases after BSA has been in contact with PS?particles,the effects being more pronounced for lower surface coverage.Furthermore,the denaturation peaks of the protein after being in contact with PS?parti-cles are broader than for BSA before adsorption. Figs.4and5compare the CD spectra of BSA before adsorption and after the exchange from silica and PS?particles,respectively.As expected for a protein that is predominantly a-helical,e.g. native BSA,the CD spectrum shows the strong negative ellipticity at222and208nm.The struc-tural elements for BSA are summarised in Table 2.The standard deviation in each one of the structural elements indicated in Table2was calcu-lated by comparing the?t obtained in separate experiments under the same experimental conditions.
Homomolecular exchange of BSA from silica surfaces does not lead to a change in the sec-ondary structure of the protein.Thus,the CD data support the DSC results that showed the same structural stability for the protein before adsorption and after exchange.On the other hand,homomolecular exchange from PS?parti-
The thermogram of BSA before adsorption is
in good agreement with those reported in the
literature obtained at similar conditions(Ru¨egg et
Table2
Denaturation temperature and enthalpy derived from DSC thermograms and secondary structure elements obtained from CD spectra of BSA,before adsorption and after being in contact with silica and PS particles(exchanged)
%Adsorbed T D(°C)D D H(J g?1)
G(mg m?2)a-Helix b-Turn
b-Sheet Unordered BSA before adsorption
57.090.71191599222913911692
00
On silica(exchanged)
10
1.156.7105823216
30
0.856.9126122215
15
3
23
58
0.6—
—
60
On PS?(exchanged)
1858.2
1.1105624416
5
24
55
9
59.4
2316
0.9
4063.0752248
0.816
W .Norde ,C .E .Giacomelli /Journal of Biotechnology 79(2000)259–268
264Fig.4.CD spectra of BSA ( )before adsorption and ( )after being in contact with silica particles.The CD spectra obtained after BSA was exchanged from silica particles at different surface coverage coincide with the one displayed on the ?gure.
4.Discussion
The primary contributions to protein adsorp-tion on a smooth,rigid surface originate from (a)electrostatic interactions between the protein and the sorbent surface,giving rise to coadsorption of small ions,(b)dispersion interaction,(c)changes in the state of hydration of the sorbent surface and parts of the protein molecule,and (d)struc-tural rearrangements in the protein (Norde and Haynes,1996;Norde,1998).These processes are not independent of each other and the magnitude and prevalence of one over the others is related to the physicochemical properties of both the partic-ular protein and sorbent surface involved.Proteins with a high internal cohesion (‘hard’proteins)have a stronger resilience against struc-tural rearrangements during adsorption while
Fig.5.CD spectra of BSA ( )before adsorption and after being exchanged from PS ?particles at different surface cover-age:( )G =1.1mg m ?2;( )G =0.9mg m ?2;(")G =0.8mg m ?2.
cles causes a structural perturbation in the BSA molecules that is more pronounced for a lower degree of surface coverage.The fraction of a -helix decreases,the b -turn content increases while the percentages of b -sheet and random coil elements are essentially the same before adsorption and after exchange.The alteration in the (secondary)structure of the exchanged BSA molecules is ac-companied by a change in the thermal stability as is demonstrated by the DSC results.Exchange of BSA from other hydrophobic sorbents such as copolypeptide and silicone surfaces also leads to a decrease in helical content (Soderquist and Wal-ton,1980).
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those that are structurally labile(‘soft’proteins) will change their structure more readily upon adsorption(Norde,1998).Both hard and soft proteins adsorb on hydrophobic surfaces because dehydration of the sorbent surface is a strong entropic driving force for adsorption.Hard proteins adsorb on hydrophilic surfaces only if they are electrostatically attracted.Soft proteins do adsorb on hydrophilic electrostatically re-pelling surfaces because the gain in conforma-tional entropy of the protein,associated with a decreased(secondary)structure(Norde and Fa-vier,1992),is suf?ciently large to overcome the electrostatic repulsion.
The initial slope of the adsorption isotherm is a measure of the interaction between protein and adsorbent,i.e.it re?ects the af?nity of the protein molecule for the sorbent surface.At this low surface coverage,where lateral interactions be-tween adsorbed molecules are negligible,the effect of the nature of the surface would be most pro-nounced.As a rule,the af?nity increases as the hydrophobicity of the surface increases although other interactions like electrostatic ones may in-terfere with this trend.Indeed,according to the adsorption isotherms presented in Fig.1,the neg-atively charged BSA does not have a high af?nity for both the negatively charged hydrophobic PS?and negatively charged hydrophilic silica surface. It demonstrates that even when electrostatic inter-action is not the determining factor in the adsorp-tion process it still has a great in?uence on the af?nity of the protein for the surface.For both negatively charged surfaces the initial part of the isotherms have similar slopes.It suggests that in the case of BSA the adsorption behaviour is not controlled by the hydrophobicity of the sorbent surface.Other literature data also indicate that BSA adsorption is mainly determined by the protein and not by the sorbent surface.For in-stance,BSA adsorption kinetics are quite similar both on hydrophobic and hydrophilic silica sur-faces(Krisdhasima et al.,1993);the a-helix con-tent of BSA decreases upon adsorption on hydrophilic(Kondo et al.,1991;Norde and Fa-vier,1992)and hydrophobic(Kondo et al.,1992) surfaces;the plateau values of the adsorption isotherms as a function of pH show more or less the same trend for very different surfaces:a bell-shaped curve with a maximum close to the isoelectric point of the protein(Kondo and Hi-gashitani,1992;Quiquampoix and Ratcliffe,1992; Haynes and Norde,1994;Yoon et al.,1996;Gia-comelli et al.,1997).These results indicate that structural rearrangements in the BSA molecule play a major role in the adsorption process;it is a direct consequence of the adaptability of BSA to changes in the environment.
The adsorption saturation value,represented by the plateau value of the isotherm,is related to the conformation of,orientation of,and lateral inter-actions between,the adsorbed molecules.BSA is a heart-shaped molecule that can be approximated by an equilateral triangle with side 8nm and depth 3nm.Hence,assuming a close packed monolayer of native molecules,the maximum ad-sorbed amount that can be achieved is around4 mg m?2.The plateau values of the adsorption isotherms in Fig.1are much lower than4mg m?2.This could be due to repulsion between the negatively charged BSA molecules and/or struc-tural relaxation,i.e.spreading,of the protein at the sorbent surface.It is remarkable that the plateau values are essentially the same for the two different sorbent surfaces.This observation may be fortuitous;in other words,it does not allow a reliable comparison to be made as to the degree of spreading and packing at the respective surfaces. It has been quite often observed that the extent of the structural change of the protein resulting from adsorption depends on the surface coverage, i.e.it varies along the adsorption isotherm.At lower protein concentration in solution,corre-sponding to lower surface coverage,the rate of arrival of the protein molecules at the sorbent surface is slower,so that the molecules are al-lowed more time to adjust their structure to the new environment before a neighbouring site be-comes occupied by subsequently arriving molecules.As a consequence,structural rear-rangements are more severe at lower surface cov-erage(Norde and Zoungrana,1998).For instance,BSA molecules adsorbed on silica parti-cles at pH7showed a strongly reduced a-helix content,which is more pronounced at lower sur-face coverage(Norde and Favier,1992).This
W.Norde,C.E.Giacomelli/Journal of Biotechnology79(2000)259–268 266
same trend was observed for BSA adsorbed on ultra?ne polystyrene particles(Kondo et al., 1992).After exchange from the sorbent surface the BSA molecules may or may not return to their original structure(as it was before adsorption).If not,the degree of structural perturbation of the exchanged molecules may depend on the surface coverage as well.
Figs.2and4and Table2show that the thermal stability and the secondary structure of BSA after being exchanged from silica particles resemble those of the protein before adsorption(native structure).This is different from BSA released from silica using morpholine as displacer(Norde et al.,1986;Norde and Favier,1992);in this case BSA recovered only a part of its original helical content.However,it should be realised that ho-momolecular exchange,on the one hand,and displacement by some other surface active molecules,on the other,are different processes which may lead to different?nal states of the protein.
It is well known that BSA adapts its conforma-tion readily and often reversibly to variations in the environmental conditions(Lin and Koenig, 1976;Carter and Ho,1994).As a consequence of pH changes albumin undergoes reversible confor-mational isomerization,as has been widely de-scribed in the literature(Peters,1985;Carter and Ho,1994).The pH dependent forms were classi?ed as the‘N’(native)form,predominant in the pH range4.5–7.0;the‘B’(basic)form occur-ring beyond pH8;the‘F’(fast migrating)form produced abruptly upon lowering the pH to B4; the‘E’(expanded)form at pH lower than3.5;and the‘A’(aged)form slowly developing at pH\8. The N–F transition involves a decrease in the content of ordered(secondary)structure.It de-creases further when the F form is changed into the E form.Accordingly,the enthalpy of thermal denaturation decreases when the pH is lowered from4.5to4and below pH3.5a thermal transi-tion is not observed anymore(Yamasaki et al., 1990).These changes were found to be reversible between pH 1.7and10.9and they involve a continuous helical-disordered transition.BSA also shows reversible changes upon heating.Lin and Koenig(1976)found that BSA remains in the native state up to42°C.Between42and50°C reversible conformational changes occur which involve a continuous increase of unordered struc-ture at the expense of the a-helix content.
It is known that adsorption on silica also re-duces the helix content of BSA,the more so the lower the surface coverage(Kondo and Higashi-tani,1992;Norde and Favier,1992),accompanied by a strong decrease in D D H(Feng and Andrade, 1993;Norde and Giacomelli,1999).After homo-molecular exchange the BSA in solution has the same(secondary)structure and the same thermal transition characteristics as before adsorption. Hence,the structural perturbations induced by the silica surface are reversible.In a way,these perturbations may be similar as those involved in the N–F(or E)transition and the thermal transi-tion between42and50°C,namely the perturbing stresses cause a decrease in helical content and a lower D D H,but the original structure is regained after releasing the stress.
When BSA is adsorbed on the PS?particles the thermogram shows no signi?cant transition,at least up to100°C(Yan et al.,1995).It could imply either complete unfolding upon adsorption or an adsorbed structure that is thermostable up to100°C.According to Kondo et al.(1992)the adsorbed BSA molecules still retain a large frac-tion of ordered(secondary)structure although less than in solution.The results presented in Figs.3and5,and Table2,show that BSA does not completely regain its structural properties af-ter exchange from the PS?surface.In other words,unlike at the hydrophilic silica surface,the adsorption induced structural rearrangements at the hydrophobic PS?surface proceed irreversibly. After exchange T D values are higher and D D H values are lower as compared to the situation before adsorption.These effects are more evident as the protein is exchanged from a surface that is less crowded with protein molecules. Irreversibility in the denaturation of BSA has also been observed upon prolonged and repeated heating and by exposure to basic conditions(Pe-ters,1985).In these cases the irreversibility has been ascribed to aggregation through intermolec-ular disulphide exchange and the formation of intermolecular b-structures.Upon adsorption
W.Norde,C.E.Giacomelli/Journal of Biotechnology79(2000)259–268267
from aqueous solution onto the PS?particles,the relatively hydrophobic core of the dissolved BSA molecule may open up in order to expose hydro-phobic amino acid residues at the hydrophobic PS?surface.Then,after release from that surface intermolecular aggregation through hydrophobic patches are likely to occur before the BSA molecules are allowed to refold into their original, native structure.The increased b-turn fraction in the exchanged BSA molecules supports this sug-gestion,as it is known that formation of b-struc-ture on the protein’s exterior is involved in intermolecular hydrogen bonding(Lin and Koenig,1976;Jakobsen and Wasacz,1990).The observation that the b-turn fraction in the ex-changed molecules increases with decreasing cov-erage of PS?surface is a further indication of aggregation after displacement from the surface. If the aggregation had taken place at the surface, the development of b-turns would have been ex-pected to be promoted by increasing the surface coverage.
The lower value of D D H after exchange may re?ect that unwinding of a-helix requires more enthalpy than the breakdown of b-turns,or it could be that aggregation hampers thermal un-folding to some extent.Furthermore,the broader transition peaks point to a larger conformational heterogeneity of the exchanged BSA molecules.
5.Conclusion
The structural characteristics of BSA molecules in aqueous solution are not affected after being exchanged from a hydrophilic silica surface.Re-lease from a hydrophobic PS?surface yields BSA molecules of which the structural properties are altered.Hence,structure rearrangements in BSA resulting from homomolecular exchange proceed reversibly at the hydrophilic surface and irre-versibly at the hydrophobic surface. Acknowledgements
This project was?nancially supported by a fellowship granted to C.E.G.by the Agricultural International Centre,Ministry of Agriculture,Na-ture Management and Fisheries,The Netherlands. References
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标准溶液配制方法
中华人民共和国国家标准 UDC543.06:54—41 GB601—88 化学试剂 滴定分析（容量分析）用标准溶液的制备 Chemicalreagent Preparationsofstandardvolumetriesolutions 1主题内容与适用范围 本标准规定了滴定分析（容量分析）用标准溶液的配制和标定方法。 本标准适用于制备准确浓度之溶液，应用于滴定法测定化学试剂的主体含量及杂质含量，也可供其他的化学产品标准选用。 2引用标准 GB603化学试剂试验方法中所用制剂及制品的制备 GB6682实验室用水规格 GB9725化学试剂电位滴定法通则 3一般规定 3.1本标准中所用的水，在没有注明其他要求时，应符合GB6682中三级水的标 准。 3.2本标准中所用试剂的纯度应在分析纯以上。 3.3工作中所用的分析天平的砝码、滴定管、容量瓶及移液管均需定期校正。3.4本标准中标定时所用的基准试剂为容量分析工作基准试剂；制备标准溶液是 所用的试剂为分析纯以上试剂。 3.5本标准中所制备的标准溶液的浓度均指20c时的浓度。在标定和使用时，如 温度有差异，应只能附录A（补充件）补正。 3.6“标定”或“比较”标准溶液浓度时，平行试验不得少于8次，两人各作4 平行，每人4平行测定结果的极差与平均值之比不得大于0.1%。两人测定结果的差值与平均值之比不得大于0.1%，最终取两人测定结果的平均值。浓度值取四位有效数字。 3.7本标准中凡规定用“标定”和“比较”两种方法测定浓度时，不得略去其中 的任何一种，且两种方法测得的浓度值之差值与平均值之比不得大于0.2%，最终以标定结果为准。 3.8制备的标准溶液与规定浓度之差不得超出规定浓度的+—5%。。 3.9配制浓度等于或低于0.02mol/L标准溶液时乙二胺四乙酸二钠标准滴定溶液 除外，应于临用前将浓度高的标准溶液用煮沸并冷却的水稀释，必要时重新标定。 3.10碘量法反应时，溶液的温度不能过高，一般在15~20c之间进行滴定。 3.11滴定分析（容量分析）用标准溶液在常温（15~25）下，保存时间一般不 得超过两个月。
各种缓冲液的配制方法
1．甘氨酸–盐酸缓冲液（0.05mol/L） 2．邻苯二甲酸–盐酸缓冲液（0.05 mol/L） 24 Na2HPO4-2H2O分子量= 178.05，0.2 mol/L溶液含35.01克/升。C4H2O7·H2O分子量= 210.14，0.1 mol/L溶液为21.01克/升。
①使用时可以每升中加入1克克酚，若最后pH值有变化，再用少量50%氢氧化钠溶液或浓盐酸调节，冰箱保存。 ② 687·H2 柠檬酸钠Na3 C6H5O7·2H2O：分子量294.12 ，0.1 mol/L溶液为29.41克/毫升。 7．磷酸盐缓冲液
2 4·2H 2Na 2HPO 4·2H 2O 分子量 = 358.22，0.2 mol/L 溶液为71.64克/升。 Na 2HPO 4·2H 2O 分子量 = 156.03，0.2 mol/L 溶液为31.21克/升。 24·2H 2KH 2PO 4分子量 = 136.09，1/15M 溶液为9.078克/升。 8．磷酸二氢钾–氢氧化钠缓冲液（0.05M ）
10．Tris –盐酸缓冲液（0.05M ，25℃） C HOCH2 NH2 分子量=121.14; 0. 1M 溶液为12.114克/升。Tris 溶液可从空气中吸收二氧化碳，使用时注意将瓶盖严。 247·H 2硼酸H 2BO 3,分子量=61.84,0.2M 溶液为12.37克/升。 硼砂易失去结晶水，必须在带塞的瓶中保存。
247·10H 2硼酸H 2BO 3，分子量=61.84, 0.2M 溶液为12.37克/升。 硼砂 易失去结晶水，必须在带塞的瓶中保存。 12．甘氨酸–氢氧化钠缓冲液（ 0.05M ） 13．硼砂-氢氧化钠缓冲液（0.05M 硼酸根） 2 47·10H 2 14．碳酸钠-碳酸氢钠缓冲液（0.1M ） 2+2+22·10H 2
蛋白质芯片的综述
蛋白质芯片的综述 摘要蛋白质芯片技术是一种高通量、微型化和自动化的蛋白质分析技术，已在多个领域得到应用,如蛋白质组学研究、新药的开发、酶与底物的相互作用和疾病检测等。论文详细介绍了蛋白质芯片技术的原理、芯片介质及蛋白质的固定技术,论述了蛋白质芯片在肿瘤研究，食品检验的应用以及传染病检测中的研究概况。分析了蛋白质芯片的问题以及应用前景。 关键词蛋白质芯片，肿瘤，食品检验，传染病检测，应用 蛋白质芯片的研究工作起始于20世纪80年代,到90年代技术日趋成熟。蛋白质芯片(protein chip)技术因具有高通量平行分析、信噪比较高、所需样品量少,以及可直接关联DNA序列和蛋白质信息等优点,自问世以来,已广泛应用于蛋白质组学、医学诊断学等领域研究,具有广阔的发展。 1.蛋白质芯片介绍 1.1 技术原理 蛋白质芯片是由固定于不同介质上的蛋白微阵列组成,这些蛋白包括抗原、抗体及标志蛋白,然后用标记的或未经标记的另外一个蛋白,如抗原、抗体或配体进行反应,有的需要经洗涤后再加入标记的二抗进行反应,从而达到放大抗原抗体反应的目的。所用的标记物有荧光物质,如Cy3(青色素,一种荧光染料)和Cy5等;酶,如辣根过氧化物酶,化学发光物质等;其他分子,如免疫金标记,然后再进行银染对反应结果显色。反应结果用扫描装置进行检测或用肉眼直接进行观察。 1.2 蛋白质芯片的介质 目前作为蛋白芯片的介质有滤膜类、凝胶类和玻璃片类,前2种介质的优点是能够保持所固定的蛋白的三维结构,但缺点是由于其质地较软,所以不能满足机械点样的强度,同时凝胶类的蛋白质芯片所点样品容易发生扩散。玻璃片的优点是成本低和性能稳定,可满足高强度的机械点样。此外,20世纪90年代中期发展的液相芯片技术使蛋白芯片技术得到进一步提高。其被喻为后基因组时代的芯片技术,也可称为灵活的多种被分析物质的检测 ( flexible multi-analyte profiling,xMAP)技术,xMAP技术是集流式技术、荧光微球、激光、数字信号处理和传统化学技术为一体的一种新型生物分子高通量检测技术,这种技术将流式检测与芯片技术有机地结合在一起,使生物芯片反应体系由固相反应改变为接近生物系统内部环境的完全液相反应体系,因此也被称为液相芯片技术[1]。 光学蛋白芯片也是新发展起来的一项技术,是将高分辨的椭偏生物传感器技术和集成化多元蛋白质芯片技术相结合发展形成的生物分子识别和检测技术。该技术的优点是无需标记待检样品,无需预处理直接检测非纯化分析物,样品用量少,检测时间短并且可以进行多元检测。 1.3 蛋白质的固定 将蛋白质固定于芯片上的方法很多,各方法的最终目的是在单位面积/体积上固定最大量的蛋白质并保持其天然构象,该环节成为蛋白质芯片技术的关键步骤之一。 蛋白质的固定可以分为两类:非专一性固定和专一性固定,非专一性固定即通过被动吸附的方式使蛋白质结合到相应的介质上,如硝酸纤维素膜和多聚赖氨酸包被的玻片通过被动吸附蛋白质的氨基或羧基来固定蛋白质,此方法产生的芯片背景值往往较高。 1. 4 蛋白质芯片的检测
论文 生物芯片技术
生物芯片技术——生物化学分析论文 08应化2 江小乔温雪燕袁伟豪张若琦 2011-5-3
一、摘要： 生物芯片技术，被喻为21世纪生命科学的支撑技术，是便携式生化分析仪器的技术核心，是90年代中期以来影响最深远的重大科技进展之一，是融微电子学、生物学、物理学、化学、计算机科学为一体的高度交叉的新技术，具有重大的基础研究价值，又具有明显的产业化前景。由于用该技术可以将极其大量的探针同时固定于支持物上，所以一次可以对大量的生物分子进行检测分析，从而解决了传统核酸印迹杂交（Southern Blotting 和Northern Blotting 等）技术复杂、自动化程度低、检测目的分子数量少、低通量(low through-put)等不足。 二、关键词 生物芯片；检测；基因 三、正文 （一）、生物芯片的简介 生物芯片技术是一种高通量检测技术，通过设计不同的探针阵列、使用特定的分析方法可使该技术具有多种不同的应用价值，如基因表达谱测定、突变检测、多态性分析、基因组文库作图及杂交测序（Sequencing by hybridization, SBH）等，为"后基因组计划"时期基因功能的研究及现代医学科学及医学诊断学的发展提供了强有力的工具，将会使新基因的发现、基因诊断、药物筛选、给药个性化等方面取得重大突破，为整个人类社会带来深刻广泛的变革。该技术被评为1998年度世界十大科技进展之一。（1）它包括基因芯片、蛋白芯片及芯片实验室三大领域。 基因芯片(Genechip)又称DNA芯片(DNAChip)。它是在基因探针的基础上研制出的，所谓基因探针只是一段人工合成的碱基序列，在探针上连接一些可检测的物质，根据碱基互补的原理，利用基因探针到基因混合物中识别特定基因。它将大量探针分子固定于支持物上，然后与标记的样品进行杂交，通过检测杂交信号的强度及分布来进行分析。 蛋白质芯片与基因芯片的基本原理相同，但它利用的不是碱基配对而是抗体与抗原结合的特异性即免疫反应来检测。蛋白质芯片构建的简化模型为：选择一种固相载体能够牢固地结合蛋白质分子(抗原或抗体)，这样形成蛋白质的微阵列，即蛋白质芯片。 芯片实验室为高度集成化的集样品制备、基因扩增、核酸标记及检测为一体
常用标准溶液的配制和标定
标准溶液的配制与标定 实训一氢氧化钠标准溶液的配制和标定 一、目的要求 1.掌握NaOH标准溶液的配制和标定。 2.掌握碱式滴定管的使用，掌握酚酞指示剂的滴定终点的判断。 二、方法原理 NaOH有很强的吸水性和吸收空气中的CO2，因而，市售NaOH中常含有Na2CO3。 反应方程式：2NaOH + CO2→Na2CO3+ H2O 由于碳酸钠的存在，对指示剂的使用影响较大，应设法除去。 除去Na2CO3最通常的方法是将NaOH先配成饱和溶液（约52%，W/W），由于Na2CO3在饱和NaOH溶液中几乎不溶解，会慢慢沉淀出来，因此，可用饱和氢氧化钠溶液，配制不含Na2CO3的NaOH溶液。待Na2CO3沉淀后，可吸取一定量的上清液，稀释至所需浓度即可。此外，用来配制NaOH溶液的蒸馏水，也应加热煮沸放冷，除去其中的CO2。 标定碱溶液的基准物质很多，常用的有草酸（H2C2O4?2H2O）、苯甲酸（C6H5COOH）和邻苯二甲酸氢钾（C6H4COOHCOOK）等。最常用的是邻苯二甲酸氢钾，滴定反应如下： C6H4COOHCOOK + NaOH →C6H4COONaCOOK + H2O 计量点时由于弱酸盐的水解，溶液呈弱碱性，应采用酚酞作为指示剂。 三、仪器和试剂 仪器：碱式滴定管（50ml）、容量瓶、锥形瓶、分析天平、台秤。 试剂：邻苯二甲酸氢钾（基准试剂）、氢氧化钠固体（A.R）、10g/L酚酞指示剂：1g酚酞溶于适量乙醇中，再稀释至100mL。 四、操作步骤 1.0.1mol/L NaOH标准溶液的配制 用小烧杯在台秤上称取120g固体NaOH，加100mL水，振摇使之溶解成饱和溶液，冷却后注入聚乙烯塑料瓶中，密闭，放置数日，澄清后备用。 准确吸取上述溶液的上层清液5.6mL到1000毫升无二氧化碳的蒸馏水中，摇匀，贴上标签。 2.0.1mol/L NaOH标准溶液的标定 将基准邻苯二甲酸氢钾加入干燥的称量瓶，于105-110℃烘至恒重，用减量法准确称取邻苯二甲酸氢钾约0.6000克，置于250 mL锥形瓶中，加50 mL无CO2蒸馏水，温热使之溶解，冷却，加酚酞指示剂2-3滴，用欲标定的0.1mol/L NaOH溶液滴定，直到溶液呈粉红色，半分钟不褪色。同时做空白试验。 要求做三个平行样品。
各种缓冲液的配制方法
24 Na2HPO4-2H2O 分子量 = 178.05，0.2 mol/L 溶液含 35.01 克/升。C4H2O7·H2O 分子量 = 210.14，0.1 mol/L 溶液为 21.01 克/升。
① 使用时可以每升中加入 1 克克酚，若最后 pH 值有变化，再用少量 50% 氢氧化钠溶液或浓盐酸调节，冰箱保存。 柠檬酸C6H8O7·H2O：分子量 210.14，0.1 mol/L溶液为 21.01 克/升。柠檬酸钠 Na3 C6H5O7·2H2O：分子量294.12，0.1 mol/L溶液为29.41 克/毫升。 22 7．磷酸盐缓冲液
242 Na2HPO4·2H2O 分子量 = 358.22，0.2 mol/L 溶液为 71.64 克/升。Na2HPO4·2H2O 分子量 = 156.03，0.2 mol/L 溶液为 31.21 克/升。 242 KH2PO4 分子量 = 136.09，1/15M 溶液为 9.078 克/升。 8．磷酸二氢钾–氢氧化钠缓冲液（0.05M） X 毫升 0.2M K2PO4 + Y 毫升 0.2N NaOH 加水稀释至 29 毫升
10．Tris–盐酸缓冲液（0.05M，25℃） 50毫升0.1M 三羟甲基氨基甲烷（Tris）溶液与X 毫升0.1N 盐酸混匀后，加水稀释至 100毫升。 C HOCH2 NH2 分子量=121.14; 0. 1M 溶液为 12.114 克/升。Tris 溶液可从空气中吸收二氧化碳，使用时注意将瓶盖严。 硼砂 Na2B4O7·H2O,分子量=381.43;0.05M 溶液（=0.2M 硼酸根）含19.07 克/升。硼酸 H2BO3,分子量 =61.84,0.2M 溶液为 12.37 克/升。硼砂易失去结晶水，必须在带塞的瓶中保存。
原子吸收标准溶液的配制
原子吸收常用的标准溶液配制方法 点击次数：1081 发布时间：2012-5-17 标准溶液的配备方法 钙元素符号-Ca 相对原子量 -40.08 仪器操作条件 波长 422.7nm 狭缝 0.4nm 灯电流 3.0毫安 燃烧器高度 8毫米 空气压力 0.3兆帕 乙炔压力 0.09兆帕 空气流量 7.0升/分 乙炔流量 1.5升/分 火焰类型氧化性兰色焰 钙Ca 标准溶液的配置 钙标准溶液浓度1000微克/毫升 称取经灼烧后的高纯氧化钙1.3992克，置于250毫升烧杯中，加入盐酸20毫升，低温加热溶解，冷却后移入1000毫升容量瓶中，用去离子水定容刻度，摇匀。此溶液1毫升=1000微克Ca。 或购置国家标准GBW(E)080261 1000微克/毫升Ca(基体5%盐酸) 标准系列与线性工作范围 配置每毫升含钙0.0， 1.0， 2.0，3.0，4.0，5.0微克2%盐酸溶液和0.2%氯化锶溶液。 钙标准使用液：吸取1毫升=1000微克钙标准溶液10.0毫升于100毫升容量瓶中，加入2毫升盐酸，用去离子水定容刻度，摇匀。此溶液1毫升=100微克钙。 氯化锶应为GR试剂 在仪器推荐条件下，标准曲线线性范围：0.0-5.0微克/毫升。 特征浓度 在仪器推荐条件下，钙的特征浓度约为：0.080微克/毫升(1%吸收)。 浓度为2微克/毫升的钙标准溶液，通常可获得0.110左右的吸光度值。 其他分析线
波长(ｎｍ) 狭缝(ｎｍ) 特征浓度之比 422.7 0.4 1.0 239.9 0.4 120 干扰及分析提示 据文献报道，在空气-乙炔焰中，铝、Be、硅、钛、钒、锆、磷酸盐、硫酸盐都会干扰钙的测定。将0.1-1%的镧或锶加进样品和标准中，能抑制上述干扰。硫酸、磷酸干扰钙的测定，测定时，样品和标准中酸的浓度应该一致，同样一份样品，酸的浓度不同所测吸光度值也不相同。要严格控制水和试剂空白，仪器喷雾系统注意防止沾污。钙有轻微的电离干扰。 试验表明，钙的吸光度与燃气和助燃气的比例、燃烧器的高度有关。在开始分析以前，应用该得标准溶液调节吸光度到最大，然后进行分析。 标准溶液的配备方法 镉元素符号-Cd 相对原子量—112.4仪器操作条件 波长228.8 nm 狭缝0.4 nm 灯电流 3.0毫安 燃烧器高度 6.5毫米 空气压力0.3兆帕 乙炔压力0.09兆帕 空气流量7.0升/分 乙炔流量 1.5升/分 火焰类型氧化性蓝色焰 镉 标准溶液的配置 镉标准溶液浓度1000微克/毫升 称取高纯镉(99.9%)0.1000克，置于250毫升烧杯中，加入10毫升盐酸，在低温电热板上加热溶解。移入100毫升容量瓶中，用去离子水定容刻度，摇匀。此溶液1毫升=1000微克镉。或购置国家标准GBW 08612 1000微克/毫升镉 (基体1%硝酸) 标准系列与线性工作范围 配置每毫升含镉0.0，0.2，0.4，0.6，0.8，1.0微克2%盐酸溶液。
2液相蛋白芯片技术
液相蛋白芯片技术 液相蛋白芯片技术由美国纳斯达克上市公司Luminex研制开发并于2 O世纪9O年代中期发展起来,就是在流式细胞技术、酶联免疫吸附试验(en zyme linked immunosorbent assay,ELISA)技术与传统芯片技术基础上开发的新一代生物芯片技术与新型蛋白质研究平台。液相蛋白芯片技术推动了功能基因组时代的蛋白质研究,相关的仪器、分析软件以及试剂盒研发备受瞩目并已形成一定的市场规模。现拟对该技术的基本原理、技术特点及其在免疫诊断与分析领域的研究与应用情况进行综合介绍。 一、液相蛋白芯片技术的基本原理 传统的蛋白芯片技术就是将蛋白质分子有序地固定在滤膜、滴定板与载玻片等固相载体上,用标记了特定荧光抗体的蛋白质等生物分子与芯片 作用,再利用荧光或激光扫描技术测定其荧光强度,通过荧光强度分析蛋白 质与蛋白质的相互作用,从而达到研究蛋白质功能或免疫诊断的目的。但固相载体难于维持蛋白质的天然构象,不利于蛋白质功能研究。 液相芯片技术在国际上被称之为xMAP(flexible MultilyteProfiling) 技术,其核心技术就是乳胶微球包被、荧光编码以及液相分子杂交。液相芯片体系以聚苯乙烯微球( beads ) 为基质,微球悬浮于液相体系,每种微球 可根据不同研究目的标定上特定抗体或受体探针,可对同一样品中多个不 同的分子同时进行检测。微球表面可进行一系列修饰以适合固定各种蛋白、
多肽或核酸等生物分子。xMAP技术可应用于蛋白或核酸的功能及其相互作用研究,分别称之为液相蛋白芯片技术与液相基因芯片技术。 液相蛋白芯片体系主要包括微球、蛋白探针分子、被检测物与报告分子四种成分。在液相系统中,为了区分不同的探针,每一种用于标记探针的微球都带有独特的色彩编码,其原理就是在微球中掺入不同比例的红色分类荧光及发色剂,可产生100种颜色差别的微球,可标记上100种探针分子,能同时对一个样品中多达100种不同目标分子进行检测。反应过程中,探针与报告分子都分别与目标分子特异性结合。结合反应结束后,使单个的微球通过检测通道,使用红、绿双色激光同时对微球上的红色分类荧光与报告分子上的绿色报告荧光进行检测,可确定所结合的检测物的种类与数量。 二、液相蛋白芯片技术的特点 液相蛋白芯片技术有机地整合了微球、激光检测技术、流体动力学、高速的数字信号处理系统与计算机运算功能,不仅检测速度极快,而且在免疫诊断以及蛋白质分子相互作用分析方面,其特异性与敏感性往往也超越常规技术。其技术特点可归纳如下。 1、反应快速,灵敏度高。反应环境为液相、微球上固定的探针与待检样品均在溶液中反应,其彼此间碰撞几率与速度相对于固相芯片或ElISA等反应模式,可增加10倍以上,因此可提高反应速度及灵敏度。抗原---抗体等蛋白
常用标准溶液配制方法
常用标准溶液配制方法
1
2一般规定 本标准中所用的水，在没有注明其他要求时，应符合GB6682中三级水的标准。 本标准中所用试剂的纯度应在分析纯以上。 工作中所用的分析天平的砝码、滴定管、容量瓶及移液管均需定期校正。 本标准中标定时所用的基准试剂为容量分析工作基准试剂；制备标准溶液是所用的试剂为分析纯以上试剂。 本标准中所制备的标准溶液的浓度均指20c 时的浓度。在标定和使用时，如温度有差异，应只能附录A（补充件）补正。 “标定”或“比较”标准溶液浓度时，平行试验不得少于8次，两人各作4平行，每人4平行测定结果的极差与平均值之比不得大于0.1%。两人测定结果的差值与平均值之比不得大于0.1%，最终取两人测定结果的平均值。浓度值取四位有效数字。 本标准中凡规定用“标定”和“比较”两种方法测定浓度时，不得略去其中的任何一种，且两种方法测得的浓度值之差值与平均值之比不得大于0.2%，最终以标定结果为准。
制备的标准溶液与规定浓度之差不得超出规定浓度的+—5%。。 配制浓度等于或低于0.02mol/L 标准溶液时乙二胺四乙酸二钠标准滴定溶液除外，应于临用前将浓度高的标准溶液用煮沸并冷却的水稀释，必要时重新标定。 碘量法反应时，溶液的温度不能过高，一般在15~20c之间进行滴定。 滴定分析（容量分析）用标准溶液在常温（15~25）下，保存时间一般不得超过两个月。 3标准溶液的制备和标定 4.1 氢氧化钠标准溶液（使用期：2个月） c(NaOH) = 1 mol/L c(NaOH) =0.5 mol/L c(NaOH) =0.1 mol/L 4.1.1 配制 称取110g氢氧化钠，溶于100ml无二氧化碳的水中，摇匀，注入聚乙烯容器中，密闭放置至溶液清亮。用塑料管吸下述规定体积的上层清夜，用无二氧化碳的水稀释至1000ml，摇匀。 c(NaOH) ，mol/L 氢氧化钠饱和溶
标准溶液的配制方法及基准物质
标准溶液的配制方法及基准物质 标准溶液是指已知准确浓度的溶液，它是滴定分析中进行定量计算的依据之一。不论采用何种滴定方法，都离不开标准溶液。因此，正确地配制标准溶液，确定其准确浓度，妥善地贮存标准溶液，都关系到滴定分析结果的准确性。配制标准溶液的方法一般有以下两种： 直接配制法 用分析天平准确地称取一定量的物质，溶于适量水后定量转入容量瓶中，稀释至标线，定容并摇匀。根据溶质的质量和容量瓶的体积计算该溶液的准确浓度。 能用于直接配制标准溶液的物质，称为基准物质或基准试剂，它也是用来确定某一溶液准确浓度的标准物质。作为基准物质必须符合下列要求： （1）试剂必须具有足够高的纯度，一般要求其纯度在%以上，所含的杂质应不影响滴定反应的准确度。 （2）物质的实际组成与它的化学式完全相符，若含有结晶水（如硼砂Na 2B 4 O 7 ?10H2O），其结晶水的数目也应与化学式完全相符。 （3）试剂应该稳定。例如，不易吸收空气中的水分和二氧化碳，不易被空气氧化，加热干燥时不易分解等。 （4）试剂最好有较大的摩尔质量，这样可以减少称量误差。常用的基准物质 有纯金属和某些纯化合物，如Cu, Zn, Al, Fe和K 2Cr 2 O 7 ，Na 2 CO 3 , MgO , K BrO 3 等，它们的含量一般在%以上，甚至可达% 。 应注意，有些高纯试剂和光谱纯试剂虽然纯度很高，但只能说明其中杂质含量很低。由于可能含有组成不定的水分和气体杂质，使其组成与化学式不一定准确相符，致使主要成分的含量可能达不到%，这时就不能用作基准物质。一些常用的基准物质及其应用范围列于表中。
表常用基准物质的干燥条件和应用
ph计标准溶液配制
中国PH计校正溶液配置的标准方法 一、引言：根据目前市场的应用情况看来，中国即我国国内使用的PH计校正的缓冲溶液有三种，即标称pH4 ，pH7 和pH9的三种缓冲溶液，分别学名为如下，笔者根据多项资料整理可得，为的是您能方便快速弄明白这些问题，详情： 1）pH4：0.05M 邻苯二甲酸氢钾溶液； 2）pH7：0.025M 磷酸二氢钾和磷酸氢二钠混合盐溶液； 3）pH9：0.01M 硼砂溶液； 接下来介绍以上3种溶液的主要配置简单方法。 二、PH计校正溶液配置的标准方法 1）pH4，邻苯二甲酸氢钾标准缓冲液： 精密称取在115±5℃干燥2～3小时的邻苯二甲酸氢钾[KHC8H4O4]10.12g，加水使溶解并稀释至1000ml。 2）pH7，磷酸盐标准缓冲液(pH7.4)： 精密称取在115±5℃干燥2～3小时的无水磷酸氢二钠4.303g与磷酸二氢钾1.179g，加水使溶解并稀释至1000ml。 另补充：磷酸盐标准缓冲液(pH6.8) 精密称取在115±5℃干燥2～3小时的无水磷酸氢二钠3.533g与磷酸二氢钾3.387g，加水使溶解并稀释至1000ml。 3）pH9，硼砂标准缓冲液：
精密称取硼砂[Na2B4O7·10H2O]3.80g(注意：避免风化)，加水使溶解并稀释至1000ml，置聚乙烯塑料瓶中，密塞，避免与空气中二氧化碳接触。 总结：从现在使用PH计来看，中国境内即国产的PH计或者是酸度计，它的校正缓冲液拥有的情况有两种： 1）即标准溶液是可以在市场上买到的，一般是在聚乙烯瓶中密闭保存的。在室温条件下标准溶液一般以保存1～2个月为宜，当发现有浑浊、发霉或沉淀现象时，不能继续使用。在4℃冰箱内存放，且用过的标准溶液不允许再倒回。 2）还可以自己买缓冲剂回去配置得。但一般厂家发货时，由于国家规定发货时有的不准有液体或药物存在，所以只能是带有的是干燥的PH缓冲剂，客户使用时需要自己配置，只要使其溶解在预先煮沸15～30分钟的去离子水中，适当冲洗试剂袋中残留的试剂。再倒入250ml容量瓶中，稀释至刻度，充分摇匀即可。 https://www.wendangku.net/doc/612872917.html,安徽诚缘科技开发有限公司专业生产PH计等相关产品
标准缓冲液的配制及常用数据.
标准缓冲液的配制及常用数据一、标准缓冲液pH值与温度对照表 二、常用缓冲溶液的配制方法 1．甘氨酸–盐酸缓冲液（0.05mol/L） 2．邻苯二甲酸–盐酸缓冲液（0.05 mol/L） X 邻苯二甲酸氢钾分子量= 204.23，0.2 mol/L邻苯二甲酸氢溶液含40.85克/升 Na2HPO4分子量= 14.98，0.2 mol/L溶液为28.40克/升。 Na2HPO4-2H2O分子量= 178.05，0.2 mol/L溶液含35.01克/升。 C4H2O7·H2O分子量= 210.14，0.1 mol/L溶液为21.01克/升。
4 ① 使用时可以每升中加入1克克酚，若最后pH 值有变化，再用少量50% 氢氧化钠溶液或浓盐酸调节，冰箱保存。 5 柠檬酸C 6H 8O 7·H 2O ：分子量210.14，0.1 mol/L 溶液为21.01克/升。 柠檬酸钠Na 3 C 6 H 5O 7·2H 2O ：分子量294.12，0.1 mol/L 溶液为29.41克/毫升。 6．乙酸–乙酸钠缓冲液（0.2 mol/L ） Na 2Ac·3H 2O 分子量 = 136.09，0.2 mol/L 溶液为27.22克/升。 7．磷酸盐缓冲液 （1 Na 2HPO 4·2H 2O 分子量 = 178.05，0.2 mol/L 溶液为85.61克/升。 Na 2HPO 4·2H 2O 分子量 = 358.22，0.2 mol/L 溶液为71.64克/升。 Na 2HPO 4·2H 2O 分子量 = 156.03，0.2 mol/L 溶液为31.21克/升。
242KH 2PO 4分子量 = 136.09，1/15M 溶液为9.078克/升。 8．磷酸二氢钾–氢氧化钠缓冲液（0.05M ） 9 巴比妥钠盐分子量=206.18;0.04M 溶液为8.25克/升 10．Tris –盐酸缓冲液（0.05M ，25℃） 50毫升0.1M 三羟甲基氨基甲烷（Tris ）溶液与X 毫升0.1N 盐酸混匀后，加水稀释至100毫升。 三羟甲基氨基甲烷（Tris ）HOCH2 CH2OH C HOCH2 NH2 分子量=121.14; 1M 溶液为12.114克/升。Tris 溶液可从空气中吸收二氧化碳，使用时注意将瓶盖严。
几种常见金属表面处理工艺
金属表面处理种类简介 电镀 镀层金属或其他不溶性材料做阳极，待镀的工件做阴极，镀层金属的阳离子在待镀工件表面被还原形成镀层。为排除其它阳离子的干扰，且使镀层均匀、牢固，需用含镀层金属阳离子的溶液做电镀液，以保持镀层金属阳离子的浓度不变。电镀的目的是在基材上镀上金属镀层，改变基材表面性质或尺寸。电镀能增强金属的抗腐蚀性(镀层金属多采用耐腐蚀的金属)、增加硬度、防止磨耗、提高导电性、润滑性、耐热性、和表面美观。 电泳 电泳是电泳涂料在阴阳两极，施加于电压作用下，带电荷涂料离子移动到阴极,并与阴极表面所产生之碱性作用形成不溶解物，沉积于工件表面。 电泳表面处理工艺的特点： 电泳漆膜具有涂层丰满、均匀、平整、光滑的优点，电泳漆膜的硬度、附着力、耐腐、冲击性能、渗透性能明显优于其它涂装工艺。电泳工艺优于其他涂装工艺。 镀锌 镀锌是指在金属、合金或者其它材料的表面镀一层锌以起美观、防锈等作用的表面处理技术。现在主要采用的方法是热镀锌。 电镀与电泳的区别 电镀就是利用电解原理在某些金属表面上镀上一薄层其它金属或合金的过程。 电泳:溶液中带电粒子（离子）在电场中移动的现象。溶液中带电粒子（离子）在电场中移动的现象。利用带电粒子在电场中移动速度不同而达到分离的技术称为电泳技术。 电泳又名——电着 (著)，泳漆，电沉积。 发黑 钢制件的表面发黑处理，也有被称之为发蓝的。其原理是将钢铁制品表面迅速氧化,使之形成致密的氧化膜保护层，提高钢件的防锈能力。 发黑处理现在常用的方法有传统的碱性加温发黑和出现较晚的常温发黑两种。但常温发黑工艺对于低碳钢的效果不太好。A3钢用碱性发黑好一些。 在高温下（约550℃）氧化成的四氧化三铁呈天蓝色，故称发蓝处理。在低温下（约3 50℃）形成的四氧化三铁呈暗黑色，故称发黑处理。在兵器制造中，常用的是发蓝处理；在工业生产中，常用的是发黑处理。 采用碱性氧化法或酸性氧化法；使金属表面形成一层氧化膜，以防止金属表面被腐蚀，此处理过程称为“发蓝”。黑色金属表面经“发蓝”处理后所形成的氧化膜，其外层主要是四氧化三铁，内层为氧化亚铁。 发蓝（发黑）的操作流程：
标准溶液配制和标定
1、氢氧化钠标准滴定溶液 1.1配制 称取110 g氢氧化钠，溶于100 ml无二氧化碳的水中，摇匀，注人聚乙烯容器中，密闭放置至溶液清亮。按表1的规定，用塑料管量取上层清液，用无二氧化碳的水稀释至1 000MI，摇匀。 表1 1.2 标定 按表 2 的规定称取于 105℃--110℃电烘箱中干燥至恒重的工作基准试剂邻苯二甲酸氢钾，加无二氧化碳的水溶解，加2滴酚酞指示液(10 g/L)，用配制好的氢氧化钠溶液滴定至溶液呈粉红色，并保持30 s。同时做空白试验。 表2 氢氧化钠标准滴定溶液的浓度〔c(NaOH)]，数值以摩尔每升(mol/ L)表示，按式(1)计算: m×1000 c(NaOH)= ------------- （ V1-V2）M 式中 : m—邻苯二甲酸氢钾的质量的准确数值，单位为克(9); V1 —氢氧化钠溶液的体积的数值，单位为毫升(mL);
V2 一空白试验氢氧化钠溶液的体积的数值，单位为毫升(mL); M一邻苯二甲酸氢钾的摩尔质量的数值，单位为克每摩尔(g/mol)【M(KHC8H4O4)= 204.22 】 2、硫酸标准滴定溶液 2.1配制 按表3的规定量取硫酸，缓缓注人1 000 mL水中，冷却，摇匀。 表3 2.2标定 按表4的规定称取于270℃—300℃高温炉中灼烧至恒重的工作基准试剂无水碳酸钠，溶于50m l.水中，加5甲基红—亚甲基蓝指示剂（或滴澳甲酚绿一甲基红指示液），用配制好的硫酸溶液滴定至溶液由绿色变为紫色（绿色变为暗红色），煮沸2 min，冷却后继续滴定至溶液再呈紫色（暗红色）。同时做空白试验。 表4 硫酸标准滴定溶液的浓度[c（1/2H2SO4）]，数值以摩尔每升(mol/L)表示 m×1000 c（1/2H2SO4）= ------------- （ V1-V2）M 式中: m—无水碳酸钠的质量的准确数值，单位为克(g); V1—硫酸溶液的体积的数值，单位为毫升(mL) ;
标准溶液的配制方法及基准物质
你标准溶液的配制方法及基准物质 2.2.1标准溶液的配制方法及基准物质 标准溶液是指已知准确浓度的溶液，它是滴定分析中进行定量计算的依据之一。不论采用何种滴定方法，都离不开标准溶液。因此，正确地配制标准溶液，确定其准确浓度，妥善地贮存标准溶液，都关系到滴定分析结果的准确性。配制标准溶液的方法一般有以下两种： 2.2.1.1直接配制法 用分析天平准确地称取一定量的物质，溶于适量水后定量转入容量瓶中，稀释至标线，定容并摇匀。根据溶质的质量和容量瓶的体积计算该溶液的准确浓度。 能用于直接配制标准溶液的物质，称为基准物质或基准试剂，它也是用来确定某一溶液准确浓度的标准物质。作为基准物质必须符合下列要求： （1）试剂必须具有足够高的纯度，一般要求其纯度在99.9%以上，所含的杂质应不影响滴定反应的准确度。
（2）物质的实际组成与它的化学式完全相符，若含有结晶水（如硼砂Na2B4O7?10H2O），其结晶水的数目也应与化学式完全相符。 （3）试剂应该稳定。例如，不易吸收空气中的水分和二氧化碳，不易被空气氧化，加热干燥时不易分解等。 （4）试剂最好有较大的摩尔质量，这样可以减少称量误差。常用的基准物质有纯金属和某些纯化合物，如Cu, Zn, Al, Fe 和K2Cr2O7，Na2CO3 , MgO , KBrO3等，它们的含量一般在99.9%以上，甚至可达99.99% 。 应注意，有些高纯试剂和光谱纯试剂虽然纯度很高，但只能说明其中杂质含量很低。由于可能含有组成不定的水分和气体杂质，使其组成与化学式不一定准确相符，致使主要成分的含量可能达不到99.9%，这时就不能用作基准物质。一些常用的基准物质及其应用范围列于表2.1中。 表2.1 常用基准物质的干燥条件和应用
常用标准溶液配制方法
中华人民共和国国家标准 UDC 543.06:54 —41 GB 601—2002 化学试剂 滴定分析（容量分析）用标准溶液的制备 Chemical reagent Preparations of standard volumetrie solutions 1主题容与适用围 本标准规定了滴定分析（容量分析）用标准溶液的配制和标定方法。 本标准适用于制备准确浓度之溶液，应用于滴定法测定化学试剂的主体含量及杂质含量，也可供其他的化学产品标准选用。 2引用标准 GB 603 化学试剂试验方法中所用制剂及制品的制备 GB 6682 实验室用水规格 GB 9725 化学试剂电位滴定法通则 3一般规定 本标准中所用的水，在没有注明其他要求时，应符合GB6682中三级水的标准。 本标准中所用试剂的纯度应在分析纯以上。 工作中所用的分析天平的砝码、滴定管、容量瓶及移液管均需定期校正。
本标准中标定时所用的基准试剂为容量分析工作基准试剂；制备标准溶液是所用的试剂为分析纯以上试剂。 本标准中所制备的标准溶液的浓度均指20c时的浓度。在标定和使用时，如温度有差异，应只能附录A（补充件）补正。 “标定”或“比较”标准溶液浓度时，平行试验不得少于8次，两人各作4平行，每人4平行测定结果的极差与平均值之比不得大于0.1%。两人测定结果的差值与平均值之比不得大于0.1%，最终取两人测定结果的平均值。浓度值取四位有效数字。 本标准中凡规定用“标定”和“比较”两种方法测定浓度时，不得略去其中的任何一种，且两种方法测得的浓度值之差值与平均值之比不得大于0.2%，最终以标定结果为准。 制备的标准溶液与规定浓度之差不得超出规定浓度的+—5%。。 配制浓度等于或低于0.02mol/L 标准溶液时乙二胺四乙酸二钠标准滴定溶液除外，应于临用前将浓度高的标准溶液用煮沸并冷却的水稀释，必要时重新标定。 碘量法反应时，溶液的温度不能过高，一般在15~20c之间进行滴定。 滴定分析（容量分析）用标准溶液在常温（15~25）下，保存时间一般不得超过两个月。 4标准溶液的制备和标定 4.1 氢氧化钠标准溶液（使用期：2个月） c(NaOH) = 1 mol/L c(NaOH) =0.5 mol/L c(NaOH) =0.1 mol/L 4.1.1 配制
标准缓冲液的配制方法样本
标准缓冲液的配制及常见数据 一、标准缓冲液pH值与温度对照表 二、常见缓冲溶液的配制方法 1．甘氨酸–盐酸缓冲液( 0.05mol/L) X毫升0.2 mol/L甘氨酸+Y毫升0.2 mol/L HCI, 再加水稀释至200毫升 甘氨酸分子量 = 75.07, 0.2 mol/L甘氨酸溶液含15.01克/升。 2．邻苯二甲酸–盐酸缓冲液( 0.05 mol/L) X毫升0.2 mol/L邻苯二甲酸氢钾 + 0.2 mol/L HCl, 再加水稀释到20毫升 邻苯二甲酸氢钾分子量 = 204.23, 0.2 mol/L邻苯二甲酸氢溶液含40.85克/升3．磷酸氢二钠–柠檬酸缓冲液
Na 2HPO 4分子量 = 14.98, 0.2 mol/L 溶液为28.40克/升。 Na 2HPO 4-2H 2O 分子量 = 178.05, 0.2 mol/L 溶液含35.01克/升。 C 4H 2O 7·H 2O 分子量 = 210.14, 0.1 mol/L 溶液为21.01克/升。 4．柠檬酸–氢氧化钠-盐酸缓冲液 ① 使用时能够每升中加入1克克酚, 若最后pH 值有变化, 再用少量50% 氢氧化钠溶 液或浓盐酸调节, 冰箱保存。 5．柠檬酸–柠檬酸钠缓冲液( 0.1 mol/L) 柠檬酸C 6H 8O 7·H 2O: 分子量210.14, 0.1 mol/L 溶液为21.01克/升。 柠檬酸钠Na 3 C 6H 5O 7·2H 2O: 分子量294.12, 0.1 mol/L 溶液为29.41克/毫升。 6．乙酸–乙酸钠缓冲液( 0.2 mol/L) Na 2Ac·3H 2O 分子量 = 136.09, 0.2 mol/L 溶液为27.22克/升。 7．磷酸盐缓冲液 ( 1) 磷酸氢二钠–磷酸二氢钠缓冲液( 0.2)
常用标准溶液的配制及标定
常用标准溶液的配制及标定 修订：段海报 一、EDTA标准溶液的配制与标定 1、EDTAD的配制 EDTA标准溶液，通常用EDTA二钠盐（乙二胺四乙酸二钠）配制。EDTA二钠盐含有两个结晶水（Na2H2Y·2H2O）,在常温度下约为含0.3%的吸附水，在80℃干燥可去吸附水，在104－14℃干燥可失去结晶水，的无水EDTA二钠盐（Na2H2Y），以上统称EDTA。Na2H2Y分子量为336.25，EDTA配制时，通常是经过标定，因此，可直接称量配制。不同浓度的EDTA标准溶液的配制可按下表进行，称取EDTA（含水），溶于5L热水中，冷却后，用脱脂棉过滤，最后稀释至60L，混匀，放置1－3天后，进行标定。 EDTA标准溶液的配制对应表 EDTA标准溶液的浓度(mol）配制60L时需要的量（g）摩尔浓度 （mol） 0.09808 2196.0 0.01 201.6 2、EDTA标准溶液的标定 标定EDTA的基准物质，在可能的情况下，尽量采用采测元素的基准物质进行。在一般情况下，多采用光谱纯金属锌作
为标定的基准。以金属锌做基准物质时，金属表面的氧化层，要用干净的剪刀刮干净，或用3M盐酸洗净，再以二次水，乙醚或丙酮洗净，在100℃烘干5min。 金属锌原子质量=65.38 锌标液的配制 将金属锌粒（99.99%以上），先用1%的硝酸浸泡除去表面上的氧化膜至光亮，然后用水冲洗三次，再用少量无水乙醇洗涤三次，在105℃烘干。（不宜过久，以免又产生氧化膜，保存于磨口瓶中）。 配制0.09808M锌标准溶液 称取12.825g的锌粒于500ml的烧杯中，加水150ml，然后分数次加入120ml（1+1）硝酸，在电热板上低温加热溶解，并彻底赶出NO2，将体积浓缩到150ml，使溶液呈无色透明。取下来冷却，移入2L容量瓶中，稀释至刻度。 Zn标准溶液的配制对应表 Zn标液的浓度（mol) 所需金属锌 的质量 理论消耗硝酸（1+1）的量 （ml） 0.09808 12.825 120.0 0.01 1.308 12.24 按照下表的取样量，移取锌标准溶液于500ml锥形瓶中，加水约80ml，加入溴酚绿指示剂（0.1%）2滴，加氨水（1+1）至溶液刚好呈蓝色，加PH=5.5的HAc-NaAc溶液15ml，二
标准溶液的配制方法
1.2 标准溶液的配制方法 化学分析大都使用溶液进行实际操作，在分析测定时又多使用标准试剂的溶液，简称标准溶液，作为分析被测元素的标准。不是什么试剂都可用来直接配制标准溶液的，必须是基准物质或标准物质才能直接配制。 (1) 基准物质 凡能用于直接配制标准溶液或标定标准溶液的物质，称为基准物质或标准物质。基准物质应符合下列要求： 1) 组成恒定，应与它的化学式完全相符，若含有结晶水，则其含量也应固定不变。如草酸(H2C2O4·2H2O)，其结晶水的含量也应与化学式完全相符。 2)纯度高，杂质的含量应少到不致于影响分析准确度，一般要求纯度99.9%以上。 3) 性质稳定，在贮存或称量过程中组成和质量不变。 4) 参与反应时应按反应式定量进行，没有副反应。 5) 应具有较大的摩尔质量，因为摩尔质量越大称量时相对误差越小。 例如，重结晶过的重铬酸钾符合上述要求，可作为基准物质，可以用来直接配制成标准溶液。但很多物质不符合上述要求，例如氢氧化钠在空气中很容易吸收空气中的二氧化碳和水分，所得的质量就不能代表纯的氢氧化钠的质量，因此氢氧化钠不是基准物质，配制成溶液，必须进行标定才能作为标准溶液。 常用的基准物质有苯甲酸、邻苯二甲酸氢钾、四硼酸钠、碳酸钠、草酸钠、重铬酸钾、氯化钠、三氧化二砷、氧化锌等，还有如银、铜、锌、镉等纯金属也可用作基准物质。 (2) 标准溶液的配制方法 1) 直接配制法：准确称取一定量的基准物质，溶解后配制成一定体积的溶液，根据物质的量和溶液的体积，即可计算出该标准溶液的准确浓度。 2) 间接配制法 (或称标定法)：有很多物质不能直接用于配制标准溶液，这时可先配制成一种近似于所需浓度的溶液，然后用基准物质 (或已经用基准物质标定过的标准溶液) 来标定它的准确浓度。 在实际工作中，有时也用“标准试样”来标定标准溶液，这样可以消除共存元素的影响。
各种化学试剂标准溶液的配制
常用试剂的配制一、标准溶液的配制 1、硫酸（H 2SO 4 ）溶液的配制： 1000mL浓度c（1/2H 2SO 4 ）=0.1mol/L，即c（H 2 SO 4 ）=0.05mol/L的硫酸溶液的配制： 取3mL左右的浓硫酸缓缓注入1000mL水中，冷却，摇匀。 新配制的硫酸需要标定，其标定方法如下： 称取于270-300℃高温炉中灼烧至恒重的工作基准试剂无水碳酸钠0.2g，溶于50mL水中，加10滴溴甲酚绿-甲基红指示液，用配制好的硫酸溶液滴定至溶液由绿色变为暗红色，煮沸2min，冷却后继续滴定至溶液再呈暗红色。同时做空白试验（取50mL水，加10滴溴甲酚绿-甲基红指示液，同样用硫酸溶液滴定至溶液由绿色变为暗红色，煮沸2min，冷却后继续滴定至溶液再呈暗红色）。计算公式为： 式中： m：无水碳酸钠的质量，g； V 1 ：滴定时所用的硫酸的体积，mL； V 2 ：空白滴定时所用的硫酸的体积，mL； M：无水硫酸钠的相对分子质量，g/mol，[M（1/2Na 2CO 3 ）=52.994）]。 测定氨氮时，氨氮含量的计算： 式中： 氨氮：氨氮含量，mg/L； V 1 ：滴定水样时所用的硫酸的体积，mL； V 2 ：空白滴定时所用的硫酸的体积，mL； M：硫酸溶液的浓度，mol/L； V：水样的体积，mL。 2、重铬酸钾（K 2Cr 2 O 7 ）溶液的配制 1000mL浓度c（1/6K 2Cr 2 O 7 ）=0.2500mol/L，即c（K 2 Cr 2 O 7 ）=0.0417mol/L的重铬酸钾溶液的配 制： 称取12.258g于120℃下干燥2h的重铬酸钾溶于水中，并移入容量瓶中，定容至1000mL，摇匀，备用。 3、硫酸亚铁铵标准溶液的配制：