当前位置：文档库 › 核磁中内标选择

核磁中内标选择

Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 645–651

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical

Analysis

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j p b

Survey and quali?cation of internal standards for quanti?cation by 1H NMR spectroscopy

Torgny Rundl?f a ,?,Marie Mathiasson a ,Somer Bekiroglu b ,1,Birgit Hakkarainen a ,b ,Tim Bowden c ,Torbj?rn Arvidsson a ,b

Medical Products Agency,P.O.Box 26,SE-75103Uppsala,Sweden

Division of Analytical Pharmaceutical Chemistry,Department of Medicinal Chemistry,Uppsala University,Biomedical Centre,Box 574,SE-75123,Uppsala,Sweden c

Division of Polymer Chemistry,Department of Materials Chemistry,Uppsala University,?ngstr?m Laboratory,Box 538,SE-75121Uppsala,Sweden

a r t i c l e i n f o Article history:

Received 23November 2009

Received in revised form 29January 2010Accepted 4February 2010

Available online 11 February 2010Keywords:

Nuclear magnetic resonance spectroscopy Quanti?cation

Internal reference standard Absolute purity

a b s t r a c t

In quantitative NMR (qNMR)selection of an appropriate internal standard proves to be crucial.In this study,25candidate compounds considered to be potent internal standards were investigated with respect to the ability of providing unique signal chemical shifts,purity,solubility,and ease of use.The 1H chemical shift (?)values,assignments,multiplicities and number of protons (for each signal),appropri-ateness (as to be used as internal standards)in four different deuterated solvents (D 2O,DMSO-d 6,CD 3OD,CDCl 3)were studied.Taking into account the properties of these 25internal standards,the most versatile eight compounds (2,4,6-triiodophenol,1,3,5-trichloro-2-nitrobenzene,3,4,5-trichloropyridine,dimethyl terephthalate,1,4-dinitrobenzene,2,3,5-triiodobenzoic acid,maleic acid and fumaric acid)were quali-?ed using both differential scanning calorimetry (DSC)and NMR spectroscopy employing highly pure acetanilide as the reference standard.The data from these two methods were compared as well as utilized in the quality assessment of the compounds as internal standards.Finally,the selected internal standards were tested and evaluated in a real case of quantitative NMR analysis of a paracetamol pharmaceutical product.

1.Introduction

Quantitative NMR (qNMR)was introduced already more than 50years ago and the ?rst study on pharmaceuticals was published in 1963[1,2].The state of the art of qNMR in the ?eld of pharmaceu-ticals and related areas was reviewed [3–6]and during the recent years scienti?c publications emerged at an escalating rate.

An advantage of qNMR compared to other instrumental analyt-ical methods is that it is a primary ratio method of measurement,since the peak areas are proportional to the number of corre-sponding nuclei giving rise to the signals [7–9].The uncertainty in quanti?cation measurement by NMR spectroscopy is low [10,11]and the performance of qNMR is similar to that of the alternative analytical techniques [12].Compared to the traditional chromato-graphic methods that are still favored in routine quantitative analyses,NMR spectroscopy have certain advantages [10]such as

?Corresponding author at:Medical Products Agency,P.O.Box 26,SE-75103Upp-sala,Sweden.Tel.:+4618174842;fax:+4618548566.

E-mail address:torgny.rundlof@mpa.se (T.Rundl?f).1

Present address:TUBITAK Marmara Research Center,Food Institute,P.O.Box 21,41470Gebze/Kocaeli,Turkey.

(i)simple and easy sample preparation,(ii)possibility to simulta-neously determine molecular structures,(iii)no need for individual experimental setup,e.g .,reference of the same compound and cal-ibrations,(iv)relatively short measuring times,(v)non-invasive and non-destructive character of the method,(vi)no need for prior isolation of the analyte present in a mixture,(vii)possibility of simultaneous quantitative analysis of multiple target analytes in a mixture.

The applicability of qNMR has increased due to availability of high sensitivity/homogeneity NMR systems together with modern software packages which allow accurate and precise data process-ing.In qNMR a reference standard of the analyte is not needed,i.e .,the quanti?cation may be performed using an internal stan-dard [1,5].Various internal standards have been used in qNMR [6],usually co-dissolved with the analyte,but also introduced in a separate coaxial insert tube [13].In recent time also quanti?ca-tion towards an electronically generated signal (peak)in the NMR spectrum,so-called Eretic,was evolved [14].However,a descrip-tive and comparative study on internal standards is non-existent in the literature.

The scope of the present work was to study compounds that can be used as internal standards using 1H NMR experiments,since 1H nuclei are at the core of qNMR applications due to high sensitivity

646T.Rundl?f et al./Journal of Pharmaceutical and Biomedical Analysis

52 (2010) 645–651

Chart 1.

and widespread presence in organic molecules,even though other NMR active nuclei like 13C,19F,and 31P can be employed [1].In qNMR the signal from the internal standard and the signal from the analyte must not overlap,and in the selection of compounds in this study attention was attempted so as to avoid normally crowded spectral regions.

In the present work 25candidate qNMR internal standards were thoroughly investigated.Chemical shift (?)values,assignments,multiplicities and number of protons (for each signal),appropri-ateness (as to be used as internal standards)of 25compounds in four different deuterated solvents (D 2O,DMSO-d 6,CD 3OD,CDCl 3)were examined.Some of these compounds were previously used in NMR quanti?cation,while some others are novel.A method for quali?cation of the standards was also developed,in order to ensure accurate and precise qNMR measurements.Quanti?cation of a pharmaceutical product was also performed using selected internal standards.2.Experimental

2.1.Materials and chemicals

Internal standard substances,whose abbreviations and cer-ti?ed purities from the manufacturer are given in respective parentheses,were as follows:dimethyl terephthalate (4,100.0%),maleic acid (5,100.0%),fumaric acid (8,100.0%),acetanilide (9,N -phenylacetamide,99.99%),dimethyl sulfone (10,99.9%),3,4,5-trimethoxybenzaldehyde (11,99.4%),hexamethylcyclotrisiloxane (12,99.9%),dimethylmalonic acid (13,99.8%),5-?uorouracil (99.9%),anthracene (99.0%),acetamide (99.9%)and l -leucine (100.0%)were purchased from Sigma Aldrich,Stockholm,Sweden,3,4,5-trichloropyridine (1,99.9%),1,3,5-trichloro-2-nitrobenzene (3,98.65%),2,4,6-triiodophenol (6,97.8%),2,3,5-triiodobenzoic acid (7,99.3%),2,4,5-trichloropyrimidine (14,99.4%),2,4,6-tri?uorobenzoic acid (99.8%),2,3,4-tri?uorobenzamide (99.9%)and 2,4,6-trichlorobenzoyl chloride (99.0%)were purchased from Fisher Scienti?c,G?teborg,Sweden,1,4-dinitrobenzene (2,99.9%)and 2,3,5-tribromothiophene (98.2%)were purchased from Alfa Aesar,Fr?lunda,Sweden,and sodium acetate (15,>99%),hexamine (99.5%),uracil (99.8%),3-sulfolene and 1,3,5-trimethoxybenzene were purchased from Merck,Stockholm,Sweden.The chemical structures of substances 1–15are depicted in Chart 1.

Disposable 5mm NMR tubes,HIP-7,as well as deuteriumoxide (D 2O,99.8%),dimethylsulfoxide-d 6(DMSO-d 6,99.9%),methanol-d 4

(CD 3OD,99.8%)and chloroform-d (CDCl 3,99.8%)with the chemical formulae and degrees of deuteration in parentheses,manufactured by Armar Chemicals,D?ttingen,Switzerland,were purchased from Glaser Lab-Kemikalier,G?teborg,Sweden.The paracetamol 500mg medicinal product was obtained from Apoteket AB,Sweden.2.2.NMR sample preparation

The NMR samples used for determination of spectral properties and T 1relaxation times were prepared by dissolving a few mil-ligrams of the respective internal standard in ca.0.8ml deuterated solvent,followed by agitation on a vibrating test tube shaker until the solutions were clear.For the compounds dissolving poorly,ca .10-min sonication treatment in ultrasonic bath was used.Finally the clear solutions were transferred into NMR tubes.

Samples for purity determination were prepared by precise weighing of 5–10mg of the selected substance and ca.5mg of acetanilide on separate aluminum pans using a Mettler-Toledo UMT2(Mettler-Toledo GmbH,Greifensee,Switzerland)microbal-ance with a resolution of 0.0001mg.Both pans were transferred to a 10ml glass sample tube,and 0.8ml deuterated solvent was added.The mixture was shaken,sonicated,and ?nally the solution was transferred into a disposable NMR tube.Six weighings were performed for each substance.

For the commercially available 500mg paracetamol product sample,one tablet was weighed and,afterwards,pounded on a mortar.Every sample was made of ca .12mg powder and 5mg of the respective internal standard.Each mixture was then dissolved in ca.1.0ml of DMSO-d 6,shaken,sonicated and the solution trans-ferred into an NMR tube.Three weighings were performed for each internal standard.2.3.NMR instrumentation

All 1H NMR spectra were acquired at 25?C using a Bruker Avance 300spectrometer operating at 300.13MHz.The spectrometer was equipped with a 5mm Z-gradient BB probe using a 1H 90?pulse width of 7.2?s.Processing and spectra handling was performed using Topspin 1.3program suite (Bruker Biospin GmbH,Rheinstet-ten,Germany).

The experimental settings were as follows:excitation frequency 6.175ppm,spectral width 20ppm,64K complex data points,mea-surement temperature 25?C,preacquisition delay (de)6?s.A 30?pulse was used.Data processing was performed using 64K data

T.Rundl?f et al./Journal of Pharmaceutical and Biomedical Analysis52 (2010) 645–651647

Table1

Spectral properties of the internal standards in different solvents.

https://www.wendangku.net/doc/e014841484.html,pound Mol.Weight(g/mol)Solvent

?H(ppm),multiplicity a,number of H b

D2O DMSO-d6CD3OD CDCl3

13,4,5-Trichloropyridine182.448.44(s)[2]c8.77(s)[2]8.61(s)[2]8.53(s)[2] 21,4-Dinitrobenzene168.118.36(s)[4]c8.46(s)[4]8.47(s)[4]8.44(s)[4] 31,3,5-Trichloro-2-nitrobenzene226.45d8.12(s)[2]7.80(s)[2]7.47(s)[2] 4Dimethyl terephthalate194.19 3.85(s)[6]c 3.89(s)[6] 3.94(s)[6] 3.96(s)[6]

8.02(s)[4]c8.09(s)[4]8.11(s)[4]8.11(s)[4]

5Maleic acid116.07 6.21(s)[2] 6.03(s)[2] 6.26(s)[2]d 62,4,6-Triiodophenol471.80d7.97(s)[2]7.97(s)[2]7.94(s)[2]

9.72(s)[1] 5.77(s)[1]

72,3,5-Triiodobenzoic acid499.817.41(d)[1]c7.72(d)[1]7.75(d)[1]7.94(d)[1]c

8.19(d)[1]c8.31(d)[1]8.35(d)[1]8.37(d)[1]c

8Fumaric acid116.07 6.73(s)[2] 6.63(s)[2] 6.76(s)[2]d

13.12(s)[2]

9Acetanilide135.16 2.05(s)[3] 2.03(s)[3] 2.12(s)[3] 2.19(s)[3]

7.15(m)[1]7.01(t)[1]7.10(t)[1]7.12(t)[1]

～7.3(m)[4]7.28(t)[2]7.30(t)[2]7.25(s)[1]

7.56(d)[2]7.53(d)[2]7.33(t)[2]

9.91(s)[1]7.51(d)[2]

10Dimethyl sulfone94.13 3.03(s)[6] 2.99(s)[6] 3.01(s)[6] 2.99(s)[6] 113,4,5-Trimethoxybenzaldehyde196.20 3.76(s)[3] 3.76(s)[3] 3.86(s)[3] 3.95(m)[9]

3.82(s)[6] 3.86(s)[6] 3.92(s)[6]7.14(s)[2]

7.19(s)[2]7.26(s)[2]7.25(s)[2]9.88(s)[1]

9.67(s)[1]9.88(s)[1]9.85(s)[1]

12Hexamethylcyclotrisiloxane222.460.06(s)[18]c0.13(s)[18]～0.1e0.18(s)[18] 132,2-Dimethylmalonic acid132.11 1.32(s)[6] 1.27(s)[6] 1.39(s)[6]d

12.61(s)[2]

142,4,5-Trichloropyrimidine183.428.67(s)[1]9.04(s)[1]8.79(s)[1]8.61(s)[1]

15Sodium acetate82.03 1.80(s)[3] 1.58(s)[3] 1.89(s)[3]d

a Splitting pattern of the signal;(s)singlet,(d)doublet,(t)triplet,(m)multiplet or overlap.

b Number of equivalent protons in the signal.

c Very limite

d solubility.

d Insoluble.

e Multiple peaks observed.

points and an exponential line broadening factor of0.3Hz.The chemical shifts were reported in parts per million(ppm)and all the spectra were referenced to the residual proton signals of the respective solvent:?H4.67for D2O,?H2.50for DMSO-d6,?H3.31 for CD3OD,?H7.27for CDCl3.

Relaxation time measurements,using the inversion recovery technique,were performed under automation using16different tau delays ranging from0.001to5s,a repetition delay of50s,8 FID repetitions,16K complex acquisition data points,8K process-ing data points,1.0Hz line broadening factor,and non-linear?t of peak intensities.

2.4.Quanti?cation by NMR

In order to ensure reliable results for the purity determinations a repetition delay of90s was used,and64FID repetitions(num-ber of scans)resulted in a suitable signal-to-noise ratio.The other experimental settings were as stated above for1H NMR spectra. The measurement time was～100min per sample.

Phasing and integration of the spectrum was performed manu-ally,and the start and end points of each integral region were forced to zero amplitude using a?fth order polynomial baseline correction algorithm.Integration regions around the signals of interest were selected to cover the entire frequency interval between the two carbon satellite signals,and did,in all cases,suf?ce a substantially wider range than the required32×the signal half width on both sides[15].Each integral edge was checked to be almost horizontal and,if needed,manual bias and/or slope correction was applied to the integral.

The area of the signals appearing on a1D1H spectrum is directly proportional to the number of protons present in the active vol-ume of the sample.Hence,using Eq.(1),all the determinations in this study were performed on1D1H NMR spectra through the proportional comparison of the peak areas integrated for both the selected signal from the internal standard and from the substance in question:

m(x)=P(std)·

MW(x)

MW(std)

nH(std)

nH(x)

m(std)

P(x)

A(x)

A(std)(1)

where m(x)and m(std)are the masses(weights)in g,MW(x)and MW(std)are the molecular weights in g/mol,P(x)and P(std)are the purities,nH(x)and nH(std)are the number of protons generating the selected signals for integration,A(x)and A(std)are the areas for the selected peaks of the analyte and the internal standard,all respectively.

2.5.Differential scanning calorimetry(DSC)

DSC measurements were performed on a Q1000DSC instrument (TA Instruments,DE,USA)equipped with a refrigerated cooling system and calibrated using an indium metal standard(99.999%). Three samples of each substance(internal standard)were weighed (1.7–2.6mg)in aluminum pans.Melting curves were recorded using encapsulated sample and empty reference pans,a sampling rate of2.0?C/min,a sampling interval of0.10s/point,and a start temperature～10?C below the melting point of the sample.Purity calculations were performed using the purity analysis function as described in TA Instruments Explorer software,that operates according to the ASTM procedure E0928[16].

648T.Rundl?f et al./Journal of Pharmaceutical and Biomedical Analysis52 (2010) 645–651

3.Results and discussion

3.1.The investigated compounds as internal standards

A suitable internal standard should meet the criteria given below:(i)ability of providing unique and stable signals(chemical shifts),(ii)available purity,(iii)solubility in different NMR solvents, (iv)easily weighable,(v)non-volatile,(vi)non-reactive,(vii)long-term stable,and(viii)having optimum molecular weight(small molecular weight compounds require very small amounts).

Facing the dif?culty of?nding a suitable internal standard for a qNMR application,there exist only limited and fragmented information in the literature.There are numerous individual studies where only single type of applications were reported [17–19],whereas papers including several internal standards are rare[5,6].Filling this important gap,the chemical shift(?) values,assignments,multiplicities and number of protons(for each signal),appropriateness of25compounds in four different deuterated solvents(D2O,DMSO-d6,CD3OD,CDCl3)were stud-ied.Some of these compounds were previously used in NMR quanti?cation,while others are novel.The criteria for the selec-tion/ranking of these compounds were chosen on the basis of needs/requirements in our laboratory.Major criteria were unique chemical shift,sharp signal(preferably one singlet),solubil-ity properties,easy-to-use(weighing).Minor selection criteria were reactivity towards the analyte/solvent(inertness),protolytic properties,purity,and toxicity.Criteria such as complexation potential,long-term stability,and extraction behavior were,how-ever,not included in the selection/ranking procedure in this study.

The suitability of the individual compounds as internal stan-dards was examined using different solvents.The spectral properties of the15highest ranked compounds were summarized (Table1),and the individual suitability of each of these compounds based on the criteria given above is discussed below.

3,4,5-Trichloropyridine(1)was readily soluble in DMSO-d6, CD3OD,and CDCl3,but almost insoluble in D2O.It was an eas-ily weighable,crystalline compound that shows1H spectra with only one singlet originating from two protons in a normally signal-free region at ca.8.6ppm.The compound is a very weak protolyte, reasonably some orders of magnitude weaker base than pyridine [20],i.e.,the1H NMR chemical shift should not be effected due to protolytic activity in the sample.

1,4-Dinitrobenzene(2)was soluble in DMSO-d6,CD3OD,CDCl3, but almost insoluble in D2O.The1H spectrum showed only one sharp singlet,originating from four protons at ca.8.4ppm.The substance is considered toxic and harmful for the environment.

1,3,5-Trichloro-2-nitrobenzene(3)was soluble in all solvents except for D2O.The molecule contains only two aromatic protons, which appeared as one sharp singlet at around8.0ppm.Notably, the chemical shift varied much between different solvents.The substance is considered toxic.

Dimethyl terephthalate(4)was easily soluble in DMSO-d6, CD3OD,and CDCl3,but almost insoluble in D2O.The proton spec-tra showed two sharp signals as singlets,one at ca.8.1ppm and another at ca.3.9ppm.The signal of the methyl group at around 3.9ppm had an unexceptional chemical shift in a normally signal-crowded area,whereas the low-?eld signal at around8.1ppm(4H) was of high potential to be employed as reference signal.

Maleic acid(5)was soluble in all solvents except CDCl3.Two sig-nals appeared in the proton spectra of maleic acid:one sharp singlet at around6.1ppm,and a broad one for the carboxylic acid protons at around11.0ppm.Although the compound is hygroscopic,maleic acid was a good choice for the quanti?cation analyses giving the credits to its distinct solubility in D2O and easy handling,i.e.,easily weighable crystalline.Maleic acid is a protolyte,i.e.,when mixed with other protolytes,the chemical shift of the signal may change to some extent.

2,4,6-Triiodophenol(6)was soluble in all solvents except for D2O. It showed a valuable reference signal at around7.95ppm,being constituted by two protons.The phenolic proton appeared at about 9.8ppm as a broad peak.The compound is a weak protolyte.

2,3,5-Triiodobenzoic acid(7)was easily soluble in DMSO-d6and CD3OD;and partly soluble in CDCl3and D2O.The proton spectra showed two doublets and one broad acidic proton signal.One of the doublets showed a unique chemical shift at around8.35ppm,suit-able for quanti?cation,whereas the broad acidic signal was ignored due to its nature.The compound is a protolyte,cf.,maleic acid above.

Fumaric acid(8)was soluble in DMSO-d6,CD3OD but not in CDCl3.It required heat and sonication to dissolve in D2O.The pro-ton spectra showed a singlet at around6.6.Only in the spectrum of fumaric acid in DMSO-d6,it was possible to observe the singlet from the carboxyl protons resonating at13.12ppm.Fumaric acid is a protolyte,cf.,maleic acid above.

Acetanilide(9)is a white to grey solid that was soluble in DMSO-d6,CD3OD and CDCl3,and sparingly soluble in D2O.It exhibited four signals and three of them were in the region where typically aromatic proton signals are found.Acetanilide was of high purity analytical grade and reasonably prized.

Dimethyl sulfone(10)was soluble in all employed solvents.How-ever,the substance was not suitable as internal standard due to low molecular weight and the fact that the signal in the spectrum comprised six protons.Probably the error in quanti?cation will be higher compared to other suitable internal standards.A signal at3.0ppm was not unique and also prone to prospective overlap problems.

3,4,5-Trimethoxybenzaldehyde(11)was soluble in DMSO-d6, CD3OD and CDCl3,and partly soluble in D2O.At?rst glance the spec-tra showed a unique aldehyde signal of one proton at ca.9.8ppm, which seemed to be advantageous.However,being an aldehyde, this compound awakened some concerns about stability and reac-tivity properties,requiring further investigations.

Hexamethylcyclotrisiloxane(12)was soluble in all solvents apart from D2O.This moisture sensitive and unstable compound had one signal at ca.0.1ppm,sporting a unique chemical shift outside of the most crowded regions.However,it contained18protons exerting a source of weight error for quanti?cation.

2,2-Dimethylmalonic acid(13)was soluble in all solvents apart from CDCl3.Two signals appear in the spectrum:1.3and12.6ppm. The methyl signal at1.3ppm was not unique and the signal of acidic protons at12.6ppm was too broad.The compound is a protolyte, cf.,maleic acid above.

2,4,5-Trichloropyrimidine(14)was soluble in DMSO-d6,CD3OD and CDCl3,and partly soluble in D2O.It showed a suitable singlet signal at around9.0ppm.The only drawback was that this com-pound is a liquid,requiring careful handling during weighing.The compound is a protolyte,cf.,3,4,5-trichloropyridine above.

Sodium acetate(15)is known to be hygroscopic.It was solu-ble in CD3OD and D2O,partly soluble in DMSO-d6but insoluble in CDCl3.Furthermore,it is well known that NMR tubes as well as other chemical lab ware may contain acetate ions as impurities, which means that a full investigation of blank samples need to be performed.The chemical shift of the signal was around1.8ppm, which was not unique as to avoiding overlap.The compound is a protolyte,cf.,maleic acid above.

The above15compounds were found to meet many of the qualities of a useful internal standard.We also studied other compounds(hexamine,uracil,l-leucine,anthracene, acetamide,2,3,4-tri?uorobenzamide,2,4,6-tri?uorobenzoic acid, 2,4,6-trichlorobenzoyl chloride,2,3,5-tribromothiophene,5-?uorouracil),that showed out not to be particularly suitable as internal standards due to various reasons, e.g.,non-unique

T.Rundl?f et al./Journal of Pharmaceutical and Biomedical Analysis 52 (2010) 645–651

649

Table 2

Purities of the selected internal standards by NMR,DSC,and as reported by the certi?cate of https://www.wendangku.net/doc/e014841484.html,pound NMR impurity peaks NMR a purity,std.dev.DSC b purity,std.dev.Certi?cate of analysis,technique 1–c 99.5±0.3%99.9±0.06%99.9%,GC 2–c

98.1±0.6%99.6±0.03%99.9%,GC 3?H 8.6,area ～1%97.8±0.1%98.0±0.05%98.7%,GC 4–c 100.0±0.3%99.9±0.02%

100.0%,GC 5–c

99.6±0.3%–d 100.0%,HPLC 6?H 1.1,area ～1%96.5±0.5%98.8±0.14%97.8%,GC 7?H 7.6,area ～2%99.8±1.2%99.5±0.03%99.3%,HPLC 8–c 99.2±0.2%

–d 100.0%,HPLC 9

–c

–d

99.9

±0.03%

100.0%e

a Weight%,n =6(n =5for compound 3).

b mol%,n =3.

c No obvious impurity peaks larger than ～0.1%detected.

d Not determined.

Analytical technique not reported in the certi?cate.

chemical shift(s),broad/heavily “splitted”signals,toxicity,and reactivity/non-stability.

Based on these observations on the aptness of the individual compounds as internal standards,compounds 1–8were stud-ied further in order to determine their absolute purity using both differential scanning calorimetry (DSC)and quantitative NMR spectroscopy.The highly pure acetanilide was employed as the reference internal standard.

3.2.Acetanilide as control reference for qNMR

In order to perform accurate quanti?cation it is essential to know the actual purity of the used internal standard.Suitable methods for determination of purity are DSC and qNMR.The performance of qNMR has been thoroughly assessed in previous studies [10,21,22].Using qNMR it is necessary to use an estab-lished reference standard of high purity, e.g .,acetanilide.The reference standard itself can advantageously be quali?ed by 1H NMR spectroscopy,with respect to observed organic impurities,in combination with measurement of the water content by,e.g .,loss-on-drying,and inorganic impurities by,e.g .,sulphated ash.In the current work,acetanilide due to its high level of purity (reported as 100.0%)and renowned stability was selected as the reference standard to be used in purity determination of the eight internal standards chosen above [23].Quanti?cation was per-formed on mixtures of acetanilide and the respective analyte in DMSO-d 6,using integrals of the acetanilide signal at 2.0ppm and the analyte signal of highest chemical shift.

In order to exemplify the employment of acetanilide in purity determination,two frequently used internal standards,3-sulfolene and 1,3,5-trimethoxybenzene,were checked for their long-term purity stabilities using the qNMR protocol described in Section

2.The compounds were more than 3years old and kept in dry environment in their original containers with tightly closed caps.The observed purity was 98.9%for 3-sulfolene,RSD 0.2%(n =6),and 99.2%for 1,3,5-trimethoxybenzene,RSD 0.2%(n =6).No obvi-ous impurity peaks,except for water,were detected in the 1H NMR spectra.The measured differential scanning calorimetry (DSC)assay at time of delivery was 99.9%for both compounds.The fact that lower assays were obtained after storage of these compounds show on the necessity to implement a quick assay check method for internal standards used in routine work.

3.3.Purities of the selected internal standards

The purities of the eight selected compounds and acetanilide were ?rst determined using DSC.The analyses performed smoothly for compounds 1,2,3,4,6,and 7as well as for acetanilide (9).Unfortunately,the analysis failed for maleic acid (5)and fumaric acid (8).The reason might be conversion of maleic to fumaric acid near the

melting point,sublimation and/or the formation of the anhydride during heating.The results from DSC analyses (Table 2)were reported as mol%.The relative standard deviation (RSD)was excellent indicating that the content was homogeneous for all com-pounds.

Purity determination by qNMR was performed for each of the eight selected internal standards using the acetanilide method described above (Table 2).

In most cases similar results were obtained using NMR and DSC,and the results also correlated well with the purities reported in the certi?cates.The fact that qNMR purities,unlike the other tech-niques,are reported as weight percentages,in?uence the reported values.High molecular weight impurities would result in lower qNMR purities as compared to,e.g .,DSC mol percentage purities.For

Fig.1.1H NMR spectrum from purity determination of dimethyl terephthalate (B)using 100%pure acetanilide (A)as a primary reference standard.The areas of the signals at 8.1ppm (B)and 2.0ppm (A)were used for purity calculation.Residual solvent peaks from DMSO-d 6and water appear at 2.5and 3.3ppm,respectively.

650T.Rundl?f et al./Journal of Pharmaceutical and Biomedical Analysis52 (2010) 645–651

low molecular weight impurities,e.g.,residual solvents,the qNMR purity would be higher than the corresponding mol percentage purity.In previous comparative purity studies,determination by the traditional mass-balance HPLC method and the1H NMR method showed similar absolute purities[24,25]for compounds with90% or higher purities.

The results(Table2)showed that compounds1,4and5were highly pure and the qNMR assays resulted in purities of99.5%, 100.0%,and99.6%,respectively,similar to the results obtained by DSC purities and the reported value in the product certi?cates.Only few tiny impurity peaks,much smaller than the13C-satellites,were present in the proton spectra.An illustrative spectrum for dimethyl terephtalate is shown in Fig.1.

Compounds2and8showed no obvious impurity peaks and the obtained qNMR purities were98.1%and99.2%,respectively,which were lower compared to purities obtained from DSC and the cer-ti?cate.The reason may be due to difference in the water content, but this was not further studied.

For compounds3and7similar results were obtained using the different techniques,but for compound6the result of the qNMR assay was lower compared to the results from the DSC and certi?-cate assays.Obvious impurity peaks of1–2area%were observed in the proton spectra of3,6and7(Table2).Compared to the other compounds,compound7showed a low precision in the qNMR purity determination.This may be due to problems with hygro-scopicity,poor solution stability,poor substance homogeneity,or other undetermined issues in sample preparation or NMR analysis, and must be considered.

3.4.Spin-lattice relaxation times(T1)

The proton spin-lattice relaxation times(T1)of the eight selected internal standards were measured on the300MHz spectrometer using a standard inversion recovery experiment[26].The resulting exponential curves have been analyzed,and the T1values of the protons of the selected compounds are reported in Table3.

Employing an insuf?ciently short delay between the repeated NMR experiments(scans)in qNMR would cause incorrect quanti?-cation https://www.wendangku.net/doc/e014841484.html,ually it is accustomed to use a minimum delay time of?ve times the longest corresponding T1value[15].The longest T111.3s was obtained for3,4,5-trichloropyridine(1)in DMSO-d6.This means that a repetition delay of57s is required in order to ensure reasonable relaxation between90?pulses.For dimethyl terephthalate(4),on the other hand,at a maximum T1Table3

1H relaxation times(T1)in seconds of the selected internal standards in various NMR solvents.

Compound a D2O(s)DMSO-d6(s)CD3OD(s)CDCl3(s)

1–b11.3 6.8 6.6

2–b 5.2 6.2 5.8

3–b10.27.37.2

4–b 1.1c,2.7d 2.4c,4.1d 2.0c,3.8d 5 6.7 2.4 4.0–b

6–b 4.4 6.1 6.8

7–b 4.5e,4.5f 3.7e,4.4f–b

810.0 4.4 5.2–b

a Concentration2–5mg/ml.

b Not determined due to low solubility.

c?H3.9–4.0.

d?H8.1.

e?H7.7–7.9.

f?H8.3–8.4.

4.1s was observed in CD3OD for the aromatic proton and,accord-ingly,a repetition delay of21s would be suf?cient.Consequently, a quickly relaxing standard would result in a shorter measurement time,assuming quick relaxation of the analyte protons.It should be noted that for a smaller pulse angle,e.g.,30?,a shorter repeti-tion delay can be used in order to maximize sensitivity in a?xed amount of instrument time[15].Since the routine qNMR method in our laboratory involves a30?pulse angle and90s repetition delay, the complete relaxation criterion between scans was ful?lled for all of these eight standards.

3.5.Employment of the selected internal standards in a real case test

Selected internal standards were used for the quanti?cation of paracetamol(acetaminophen,N-(4-hydroxyphenyl)acetamide)in a500mg paracetamol tablet.All samples were portions taken from pulverized material from one and single tablet,DMSO-d

was used

in order to ensure maximum recovery of paracetamol and the inter-

nal standard,and quanti?cation was made by integration of the

methyl signal of paracetamol at ca.2.0ppm and a non-overlapping

signal of the respective internal standard.A typical example spec-

trum is shown in Fig.2.

Different purities of the standards obtained by NMR and DSC

and from the certi?cate of analysis,reported in Table2above,were

used for the assay calculations.The results are reported in Table4.

Fig.2.Representative1H NMR spectrum from the determination of paracetamol(A)content in a paracetamol500mg tablet,using dimethyl terephthalate(B)as internal standard.The areas of the signals at8.1ppm(B)and2.0ppm(A)were used for content calculation.Residual solvent peaks from DMSO-d6and water appear at2.5and3.3ppm, respectively.

T.Rundl?f et al./Journal of Pharmaceutical and Biomedical Analysis52 (2010) 645–651651

Table4

Determination of paracetamol content in a500mg paracetamol tablet in DMSO-d6 using different internal standards,for which purity was determined by different methods as reported in Table2.

Internal standard used Paracetamol content(mg)

NMR a DSC b Certi?cate c RSD d

1517519519 1.4% 2518526528 1.0% 3527528532 2.9% 45175175170.7% 5514–e5160.4% 65155275220.9% 7519518517 1.3%

a Content calculated using the NMR purity of the standard.

b Content calculated using the DSC purity of the standard.

c Content calculate

d using th

e certi?cate purity o

f the standard.

d Thre

e weighings were performed for each determination.

e Not determined.

The assay of paracetamol fell well within the limits for a sin-gle tablet determination[27],and varied from514mg/tablet for maleic acid using the NMR purity of the standard,to532mg/tablet for1,3,5-trichloro-2-nitrobenzene using the certi?cate purity.The latter compound showed the largest measurement uncertainty among the studied standards(RSD2.9%,n=3).

The fact that different standards produce different results may be due to uncertain purity,which in turn emphasizes the need for proper quali?cation of the standard prior to utilization.The lowest,as well as most homogenous paracetamol assays were obtained when the purity of the respective standard was certi-?ed by NMR spectroscopy(514–527mg/tablet;Table4).Somewhat higher content of paracetamol and lower precision were observed using DSC purities(517–528mg/tablet)and certi?cate purities (516–532mg/tablet).In summary,a slight better homogeneity and, in general,a somewhat lower paracetamol content was obtained when the NMR purity was used for calculation.An NMR spectrum would also add information,such as chemical shifts and magni-tudes,about possible impurity peaks that may interfere within the integration region of the analyte.If no NMR purity is available,it is recommended that the qNMR internal standard to be used is more than99%pure,in order to achieve maximum accuracy.

4.Conclusion

In this work,a series of25ordinary organic compounds were studied for their aptness as internal standards.Out of these, eight internal standards(2,4,6-triiodophenol,1,3,5-trichloro-2-nitrobenzene,3,4,5-trichloropyridine,dimethyl terephthalate, 1,4-dinitrobenzene,2,3,5-triiodobenzoic acid,maleic acid and fumaric acid)were tested and considered to be suitable for routine qNMR applications.None of these internal standards is an ideal or universal standard,but the availability of this set of standards would enable the quanti?cation of almost all prospective drug(or similar)samples requested,since these eight compounds repre-sent a broad diversity both in providing a comprehensive range of different chemical shifts and in supplying the necessary physi-cal properties including solubility in four different common NMR solvents,i.e.,D2O,DMSO-d6,CD3OD,CDCl3.

Purity determination of the standards was easily performed using highly pure acetanilide as a qNMR control reference.More-over,characterization by NMR and DSC in comparison to the certi?cated purities,and application in quanti?cation of a parac-etamol product,yielded insight into the appropriateness of the standards for applicability in qNMR.It was evident that careful purity/impurity control of the standard by NMR is to be recom-mended instead of characterization by other techniques of analysis. References

[1]U.Holzgrabe,I.Wawer,B.Diehl,NMR Spectroscopy in Pharmaceutical Analysis,

?rst ed.,Elsevier,Amsterdam,2008.

[2]D.P.Hollis,Quantitative analysis of aspirin,phenacetin,and caffeine mix-

tures by nuclear magnetic resonance spectrometry,Anal.Chem.35(1963) 1682–1684.

[3]D.M.Rackham,Recent applications of quantitative nuclear magnetic resonance

spectroscopy in pharmaceutical research,Talanta23(1976)269–274.

[4]B.W.K.Diehl,F.Malz,U.Holzgrabe,Quantitative NMR spectroscopy in the qual-

ity evaluation of active pharmaceutical ingredients and excipients,Spectrosc.

Europe19(2007)15–19.

[5]V.Rizzo,V.Pinciroli,Quantitative NMR in synthetic and combinatorial chem-

istry,J.Pharm.Biomed.Anal.38(2005)851–857.

[6]G.F.Pauli,B.U.Jaki,https://www.wendangku.net/doc/e014841484.html,nkin,Quantitative1H NMR:development and poten-

tial of a method for natural products analysis,J.Nat.Prod.68(2005)133–149.

[7]H.Jancke,NMR als prim?re analytische me?methode,https://www.wendangku.net/doc/e014841484.html,b.

46(1998)720–722.

[8]B.King,The practical realization of the traceability of chemical measurements

standards,Accred.Qual.Assur.5(2000)429–436.

[9]H.Jancke,F.Malz,W.Haesselbarth,Structure analytical methods for quantita-

tive reference applications,Accred.Qual.Assur.10(2005)421–429.

[10]F.Malz,H.Jancke,Validation of quantitative NMR,J.Pharm.Biomed.Anal.38

(2005)813–823.

[11]G.Maniara,K.Rajamoorthi,S.Rajan,G.W.Stockton,Method performance and

validation for quantitative analysis by1H and31P NMR spectroscopy.Applica-tions to analytical standards and agricultural chemicals,Anal.Chem.70(1998) 4921–4928.

[12]R.J.Wells,J.M.Hook,T.S.Al-Deen,D.B.Hibbert,Quantitative nuclear mag-

netic resonance(QNMR)spectroscopy for assessing the purity of technical grade agrochemicals:2,4-dichlorophenoxyacetic acid(2,4-D)and sodium 2,2-dichloropropionate(Dalapon sodium),J.Agric.Food.Chem.50(2002) 3366–3374.

[13]T.J.Henderson,Quantitative NMR spectroscopy using coaxial inserts containing

a reference standard:purity determinations for military nerve agents,Anal.

Chem.74(2002)191–198.

[14]V.Silvestre,S.Goupry,M.Trierweiler,R.Robins,S.Akoka,Determination of

substrate and product concentrations in lactic acid bacterial fermentations by proton NMR using the ERETIC method,Anal.Chem.73(2001)1862–1868. [15]D.L.Rabenstein,D.A.Keire,Quantitative chemical analysis by NMR,Practical

Spectrosc.11(1991)323–369.

[16]ASTM E928-08:Standard test method for determination of purity by differen-

tial scanning calorimetry,ASTM International,West Conshohocken,PA,USA, 2008.

[17]N.G.G?ger,H.K.Parlatan,H.Basan,A.Berkkan,T.?zden,Quantitative determi-

nation of azathioprine in tablets by1H NMR spectroscopy,J.Pharm.Biomed.

Anal.21(1999)685–689.

[18]A.A.Salem,H.A.Mossa,B.N.Barsoum,Application of nuclear magnetic res-

onance spectroscopy for quantitative analysis of miconazole,metronidazole and sulfamethoxazole in pharmaceutical and urine samples,J.Pharm.Biomed.

Anal.41(2006)654–661.

[19]G.M.Hanna,https://www.wendangku.net/doc/e014841484.html,u-Cam,A stability-indicating proton nuclear magnetic res-

onance spectroscopic method for the analysis of propantheline bromide in pharmaceutical samples,Pharmazie56(2001)700–703.

[20]C.-E.Lin,C.-C.Chen,H.-W.Chen,H.-C.Huang,C.-H.Lin,Y.-C.Liu,Optimization of

separation and migration behavior of chloropyridines in micellar electrokinetic chromatography,J.Chromatogr.A910(2001)165–171.

[21]L.Grif?ths,A.M.Irving,Assay by nuclear magnetic resonance spectroscopy:

quanti?cation limits,Analyst123(1998)1061–1068.

[22]S.Bekiroglu,O.Myrberg,K.?stman,M.Ek,T.Arvidsson,T.Rundl?f, B.

Hakkarainen,Validation of a quantitative NMR method for suspected coun-terfeit products exempli?ed on determination of benzethonium chloride in grapefruit seed extracts,J.Pharm.Biomed.Anal.47(2008)958–961.

[23]V.Pinciroli,R.Biancardi,N.Colombo,M.Colombo,V.Rizzo,Characterization

of small combinatorial chemistry libraries by1H NMR.Quantitation with a convenient and novel internal standard,https://www.wendangku.net/doc/e014841484.html,b.Chem.3(2001)434–440. [24]V.Pinciroli,R.Biancardi,G.Visentin,V.Rizzo,The well-characterized synthetic

molecule:a role for quantitative1H NMR,Org.Process.Res.Dev.8(2004) 381–384.

[25]S.-Y.Liu,C.-Q.Hu,A comparative uncertainty study of the calibration of

macrolide antibiotic reference standards using quantitative nuclear magnetic resonance and mass balance methods,Anal.Chim.Acta602(2007)114–121.

[26]R.L.Vold,J.S.Waugh,M.P.Klein,D.E.Phelps,Measurement of spin relaxation

in complex systems,J.Chem.Phys.48(1968)3831–3832.

[27]General Chapter20906:Uniformity of content of single-dose preparations,

European Pharmacopeia6,Council of Europe,Strasbourg,France,2008.

参照物选择的几个原则

参照物选择的几个原则泰州市高港实验学校蒋长春我们要描述一个物体是否运动或怎样运动时，必须要事先选择一个假定为不动的物体作为研究对象参照的标准，这个标准就是参照物．只有选定了参照物，我们才可以假想自己就站在参照物上去观察，也才能确定其它物体的运动状态．那么，为了描述物体的运动，我们该如何选择参照物呢？从运动学角度看，参照物可以任意选择，而且所选的参照物都是平权的，但对于同一个物体运动状态的研究，选择不同的物体作为参照物，往往描述的运动情况不同．对一个具体的运动学问题，我们要根据实际情况去选择合适的物体作为参照物，通常我们从以下四个角度进行选择．一、“一般性”原则．所谓“一般性”原则是指在描述地面上以及地面附近的物体运动状态时，一般选择地面作为参照物，或默认地面为参照物．我们生活在地面上，日常描述某个物体运动状态时，其实早已把地面作为“自然参照物”，而自己并没有意识到，比如“骏马奔驰”、“飞机起飞”、“滚滚长江东逝水”、“北风吹，雁雪纷纷”、“太阳东升西落”、“巍巍青山，岿然不动”、“地球同步卫星”等等．对我们来说，选择地面作为参照物，描述地面上以及地面附近的大多数物体的运动状态时，更符合我们的日常认知．例1如图所示，电影《闪闪的红星》主题歌的前两句歌词是：“小小竹排江中游，巍巍青山两岸走”，歌词中的“竹排”是以为参照物的，“青山”是以为参照物的．解析：本题中涉及到竹排、青山等物体．在第一句话中，研究对象是竹排，若以另一物体青山作标准，竹排是运动的（江中游），所以青山是参照物；在第二句话中，研究对象是青山，若以另一物体竹排作标准，青山是运动的（两岸走），所以竹排是参照物．我们一般认为竹排运动是理所当然的，因为竹排选择的参照物是地面；而说青山运动，有点匪夷所思，因为青山选择的参照物不是地面而是竹排，不符合我们的日常认知．如果都选地面作为参照物，那么竹排是运动的、青山是静止的．歌词中，只不过是词作者巧妙地将“青山”与“竹排”互为参照物，

归一化法、外标法、内标法的区别

色谱定量方法一、归一化法由于组分的量与其峰面积成正比，如果样品中所有组分都能产生信号，得到相应的色谱峰，那么可以用如下归一化公式计算各组分的含量。 (7.34) 若样品中各组分的校正因子相近，可将校正因子消去，直接用峰面积归一化进行计算。中国药典用不加校正因子的面积归一化法测定药物中各杂质及杂质的总量限度。 (7.35) 归一化法的优点是：简便、准确、定量结果与进样量重复性无关(在色谱柱不超载的范围内)、操作条件略有变化时对结果影响较小。缺点是：必须所有组分在一个分析周期内都流出色谱柱，而且检测器对它们都产生信号。不适于微量杂质的含量测定。二、外标法用待测组分的纯品作对照物质，以对照物质和样品中待测组分的响应信号相比较进行定量的方法称为外标法。此法可分为工作曲线法及外标一点法等。工作曲线法是用对照物

质配制一系列浓度的对照品溶液确定工作曲线，求出斜率、截距。在完全相同的条件下，准确进样与对照品溶液相同体积的样品溶液，根据待测组分的信号，从标准曲线上查出其浓度，或用回归方程计算，工作曲线法也可以用外标二点法代替。通常截距应为零，若不等于零说明存在系统误差。工作曲线的截距为零时，可用外标一点法(直接比较法)定量。外标一点法是用一种浓度的对照品溶液对比测定样品溶液中i组分的含量。将对照品溶液与样品溶液在相同条件下多次进样，测得峰面积的平均值，用下式计算样品中i组分的量： W＝A(W)／(A)(7.36) 式中W与A分别代表在样品溶液进样体积中所含i 组分的重量及相应的峰面积。(W)及(A)分别代表在对照品溶液进样体积中含纯品i组分的重量及相应峰面积。外标法方法简便，不需用校正因子，不论样品中其他组分是否出峰，均可对待测组分定量。但此法的准确性受进样重复性和实验条件稳定性的影响。此外，为了降低外标一点法的实验误差，应尽量使配制的对照品溶液的浓度与样品中组分的浓度相近。三、内标法选择样品中不含有的纯物质作为对照物质加入待测样品溶液中，以待测组分和对照物质的响应信号对比，测定待

参照物题目练习

1.物理学中把一个物体相对于另一个物体位置的改变称为机械运动，这里所说的另一个物体，即事先选定的标准物体，叫做参照物。 2.“神舟六号”飞船升空时，以固定在飞船外的摄像头为参照物，飞船是静止；以地球为参照物，飞船是运动。 3.加油机给战斗机加油，如图3-20所示，以加油机为参照物，战斗机是_静止____的（填 “静止”或“运动”）如果战斗机在３s 内飞行了0.6km,则它的速度是_______200________m/s 4.观察图3-21甲可知汽车做匀速直线运动；观察苹果下落时的频闪照片（图 3-21乙），可知苹果做变速直线运动。 2.在电视连续剧《西游记》里，常常有孙悟空“腾云驾雾”的镜头，这通常是采用“背景拍摄法”：让“孙悟空”站在平台上，做着飞行的动作，在他的背后展现出蓝天和急速飘动的白云，同时加上烟雾的效果；摄影师把人物动作和飘动的白云及下面的烟雾等一起摄入镜头。放映时，观众就感觉到孙悟空在腾云驾雾。这里，观众所选的参照物是（ C ） A ．孙悟空 B ．平台 C ．飘动的白云 D ．烟雾 3.明代诗人曾写下这样一首诗：“空手把锄头，步行骑水牛，人在桥上走，桥流水不流” 其中“桥流水不流”之句应理解成其选择的参照物是（ A ） A ．水 B ．桥 C ．人 D ．地面 4．机械运动是一种常见的运动,下列关于机械运动的说法中，正确的是（ B ） A ．物体是运动还是静止，与参照物的选择无关，对任何参照物而言结果都是相同的 B ．所说的参照物就是假定静止不动的物体，仅是以它作为研究其它物体运动的标准 C ．自然界无所谓运动和静止 D ．在研究物体的运动时，往往以地球为参照物，因为地球是静止不动的 2．为了实现全球快速、简捷地通信，人类发射了各种各样的通信卫星，同步通信卫星是其中最重要的一种．同步通信卫星：（ B ） A ．在高空静止不动 B. 相对于地球静止 C ．相对于月亮静止 D ．相对于太阳静止 5．判断下述几个运动以地面为参照物的是 ( A ) A 、太阳从东方升起 B 、月亮躲进云里 C 、客车里的乘客看到路旁的树木向后退 D 、飞机里的飞行员看到大地向北运动 8．袋鼠妈妈把小袋鼠放在育儿袋中后，在草地上跃进．相对于___地面___，它们都在运动，相对于_ 袋鼠妈妈_____，小袋鼠是静止的．图 3-20 图3-21

制剂分析外标法,内标法与混标法

书P83例7（内标法）：本品每1g含樟脑164mg，P86例8（外标法），P86例9本品每1g含黄芪甲苷为0.2mg，供试品制备取样量为1g.（对数方程外标两点法）可参考P231-233，6个验证内容。小儿热速清口服液中黄芩苷的含量测定(外标一点法)：（1）对照品溶液的制备：取黄芩苷对照品约10mg，精密称定，置200ml量瓶中，加50%甲醇适量，置热水浴中振摇使溶解，放冷，加50%甲醇至刻度，摇匀，即得。（实际浓度为0.0504mg/ml) （2）供试品溶液的制备：精密量取本品0.5ml，置D101大孔吸附树脂柱（内径约1.5cm，柱高10cm）上，用70ml水，以流量1.5ml/min洗涤，继续用40%乙醇洗脱，弃去7~9ml，收集续洗脱液，置50ml量瓶中，定容至刻度，摇匀，即得。（3）标准曲线的绘制：精密吸取浓度为0.0504mg/ml的黄芩苷对照品溶液1.0ml，2.0ml，5.0ml，8.0ml，10.0ml，分别置10ml的量瓶中，用甲醇稀释至刻度，摇匀。精密吸取上述对照液10ul注入液相色谱仪，记录峰面积。以进样量X（ug/ml）对峰面积Y进行回归处理，得回归方程Y=25.540x-0.1061（r=1.0000），结果见表1，表明黄芩苷进样量5.04~50.4ug/ml范围内与峰面积呈良好的线性关系，如图。表1 黄芩苷对照品的线性关系考察进样量(ug/ml) 峰面积回归方程相关系数 5.04 128.7 10.08 257.5 25.20 643.1 Y=25.540x-0.1061 r=1.0000 40.32 1029.6 50.40 1287.3 （4）专属性试验：在与样品测定完全相同的条件下，分别精密吸取对照品溶液，供试品溶液及阴性对照液各10ul，进样分析，观察色谱图。如图2，阴性无干扰，说明该实验方法的专属性较强。

色谱分析中归一化法、外标法、内标法的区别

色谱分析中面积归一化法、外标法、内标法适用范围及优缺点简介在色谱分析中，即我们常用的高效液相色谱分析（HPLC）和气相色谱分析（GC）分析中，进行分析时，通常采用三种方法：面积归一化法、外标法、内标法。这三种方法的适用范围及各自的优缺点是什么呢？在这里简单做一介绍。 1、归一化法。即在一定分析条件下，样品经过直接溶解，过滤等操作以后,直接进分析仪器检测，得到色谱图。通常用于粗略检查样品中的各出峰成分含量，用于定性和粗略的定量。优点：与进样量准确度无关、与仪器和分析条件有关。缺点：a.在此条件之下，所有有效组分必须出峰，且所有组分必须在一个分析周期内流出色谱峰； b.定量计算必须先知道各成分的校正因子，校正因子的求出较麻烦。 2、易挥发性的油脂类化合物和混合性气体、液体，可用GC归一化法进行定量检测。例如食用油里面各成分的含量测定。 2、外标法。用待测组分的纯品作为对照物质，以对照物质和样品中待测组分的响应信号（即峰面积大小）相比较进行定量的方法。优点：简便；只关注待测成分出峰，不需要所有成分出峰。缺点：a.必须有被测组分的纯品作为标准对照物； b.此方法准确性受进样重复性和实验条件稳定性的影响。 3、内标法。选择样品中不含有的纯物质作为内标物加入待测样

品中，以待测组分和内标物的响应信号（即峰面积大小）对比，对待测组分定量的方法。优点：a.是一种比较准确的定量方法； b.定量结果与进样量重复性无关（在色谱柱不超载范围内）； c.只需要内标物与被测物出峰，达到一定的分离度即可； d.常用于样品的GC定量检测以及微量成分含量检测；缺点：配置较麻烦；内标物需要跟待测组分在同样条件下出峰，且分离度较好，所以选择合适的内标物比较困难。

人力资源管理师(四级)教材中提到的原则和方法

第一章人力资源规划企业组织信息的特点：社会性、流动性、不规则性、连续性、浓缩性、替代性企业组织信息采集和处理的基本原则： 1、准确性原则 2、系统性原则 3、针对性原则 4、及时性原则 5、适用性原则 6、经济性原则企业组织信息采集的方法（一）档案记录法（二）调查研究法 1、询问法（1）当面询问法。适用：采集内容比较复杂，要求比较细致的信息。（2）电话调查法. 适用：采集一些简单信息。（3）会议调查询问法。适用：学者、专家或企业高层人士（4）邮寄调查法。适用：采集内容比较简单，答题要求不高，时限较长的调查。（5）问卷调查法。适用：费用适中，回收率较高，效果良好。 2、观察法（1）直接观察法。适用：采集内容复杂多变，被调查者比较集中，被调查内容固定但调查地点可变的调查。（2）行为记录法。适用：采集内容复杂多变，被调查者比较集中，调查地点固定的调查。第一单元劳动定额的基本形式劳动定额的种类（一）按劳动定额的表现形式分类 1、时间定额 2、产量定额 3、看管定额 4、服务定额 5、工作定额 6、人员定额 7、其他形式的劳动定额（二）按劳动定额的实施范围分类 1、统一定额 2、企业定额 3、一次性定额（三）按劳动定额的用途分类 1、现行定额 2、计划定额 3、设计定额 4、不变定额（四）按劳动定额编制的综合程度分类 1、时间定额 2、产量定额（五）按劳动定额的制定方法分类 1、经验估工定额 2、统计定额 3、技术定额 4、类推比较定额（六）按照劳动定额水平的高低分类 1、先进定额 2、平均先进或先进合理的定额 3、落后定额第二单元劳动定额及其管理制度制定劳动定额的基本方法 1、经验估工法 2、统计分析法（重点看下面） 3、类推比较法：做法：（1）把产品结构和工艺相同的零件或工序进行分组排列，在各组中选择具有代表性的典型零件，并根据直径、长度、精度、重量等影响工时消耗的因素，按照工序制定出典型定额标准。（2）根据典型定额来类推比较，制定同类型相似零件的工序定额。 4、技术定额法。步骤：1、分解工序；2、分析设备状况；3、分析生产组织与劳动组织；4现场观察和分析计算。第一单元工作岗位调查方式工作岗位研究的特点：1、对象性 2、系统性 3、综合性 4、应用性 5、科学性工作岗位研究的原则 1、系统的原则。任何一个系统都具有以下四个基本特征：1、整体性； 2、目的性； 3、相关性； 4、环境适应性 2、能级的原则。工作岗位能级从高到底，可区分四大层次：决策层、管理层、执行层和操作层。 3、标准化原则。标准化表现为简化、统一化、通用化、系统化等多中形式和方法。 4、最优化原则。从中优选出成本费用底、效用信度较高的方法。

仪器分析作业题外标法内标法

一、外标一点法【含量测定】芍药苷照高效液相色谱法（附录Ⅵ D)测定。色谱条件与系统适用性试验以十八烷基硅烷键合硅胶为填充剂；以甲醇／L 磷酸二氢钾溶液(40：65)为流动相；检测波长为230nm 。理论板数按芍药苷峰计算应不低于3000。对照品溶液的制备取经五氧化二磷减压干燥器中干燥36小时的芍药苷对照品适量，精密称定，加甲醇制成每1ml 含的溶液，即得。供试品溶液的制备取本品粗粉约，精密称定，置具塞锥形瓶中，精密加入甲醇25ml ，称定重量，浸泡4小时，超声处理20分钟，放冷，再称定重量，用甲醇补足减失的重量，摇匀，滤过，取续滤液，即得。测定法分别精密吸取对照品溶液与供试品溶液各10μl，注入液相色谱仪，测定，即得。本品含芍药苷(C 23H 28O 11)不得少于％。解：设测得对照品溶液和供试品溶液峰面积为A 对=550000 A 样=7800 已知对照品溶液浓度C 对=ml ml /mg 0071.0ml /mg 5.0550000 7800)(===）（对对样样C A A C m g 5.177l 101000 m l 25l 10m l /m g 0071.0V C m =?? ?==μμ样样样供试品中芍药苷的含量 X%= %5.35%1001000 g 5.0m g 5.177=?? 药典规定药材含芍药苷(C 23H 28O 11)不得少于％，供试品中芍药苷的含量为%，故供试品药材合格。

二、外标两点法【含量测定】黄芪甲苷照高效液相色谱法（附录Ⅵ D）测定。色谱条件与系统适用性试验以十八烷基硅烷键合硅胶为填充剂；以乙腈-水(32：68)为流动相；蒸发光散射检测器检测。理论板数按黄芪甲苷峰计算应不低于4000。对照品溶液的制备取黄芪甲苷对照品适量，精密称定，加甲醇制成每1ml 含的溶液，即得。供试品溶液的制备取本品中粉约4g ，精密称定，置索氏提取器中，加甲醇40ml ，冷浸过夜，再加甲醇适量，加热回流4小时，提取液回收溶剂并浓缩至干，残渣加水10ml ，微热使溶解，用水饱和的正丁醇振摇提取4次，每次40ml ，合并正丁醇液，用氨试液充分洗涤2次，每次40ml ，弃去氨液，正丁醇液蒸干，残渣加水5ml 使溶解，放冷，通过D101型大孔吸附树脂柱(内径为，柱高为12cm)，以水50ml 洗脱，弃去水液，再用40％乙醇30ml 洗脱，弃去洗脱液，继用70％乙醇80ml 洗脱，收集洗脱液，蒸干，残渣加甲醇溶解，转移至5ml 量瓶中，加甲醇至刻度，摇匀，即得。测定法分别精密吸取对照品溶液10μl、20μl，供试品溶液20μl，注入液相色谱仪，测定，用外标两点法对数方程计算，即得。本品按干燥品计算，含黄芪甲苷(C 41H 68O 14)不得少于％。解：设测得对照品溶液色谱峰面积分别为A 1=259457 A 2=523039，测得供试品溶液色谱峰面积A 3=2983。已知对照品浓度C 对=ml ，对照品进样量 g 5mg 005.0l 10ml /mg 5.0m 1μμ==?= g 10mg 01.0l 20ml /mg 5.0m 2μμ==?= 根据对照品峰面积和进样量可得如下图所示方程即y=+5x-4125，得a=，b=5

人教版八年级物理上册第一章机械运动与参照物的选择练习题及参考答案

机械运动与参照物的选择练习题一、填空题（35分） 1.物理学把______________________________叫做机械运动。 2.在研究物体做机械运动时，被选来作为标准的物体叫，同一个物体是运动还是静止的.取决于。这就是运动与静止的相对性. 3.我国古书《套买曜》上记载有：“人在舟中闭牖（门窗）而坐，舟行而人不觉”这是运动的的生动描述，其中“舟行”是以为参照物，“人不觉”是以为参照物. 4.乘客坐在行驶的火车上，以火车为参照物人是的，以路旁的树木为参照物时，人是. 5.平时所说“月亮躲进云里”是以为参照物，说“乌云遮住了月亮”是以为参照物.我国发射的风云二号通讯卫星相对是静止的.相对于是运动的.人们常说地球在公转的同时自转着，“公转”是以为参照物，自转又是以为参照物. 6.空中加油机在空中给歼击机加油的过程中，若以为参照物，他们都是运动的，若以加油机为参照物，歼击机是的. 7.“旭日东升”是以为参照物的，“日落西山”是以为参照物的. 8.坐在甲车里的人，看见路边树木向北运动，他是以为参照物.他看到并排的乙车静止，若以树为参照物，乙车是的. 9.长征三号火箭运载同步卫星升空，此时，以地球为参照物，卫星是的，以火箭为参照物，卫星是的，当卫星脱离火箭绕地球运转时，以地球为参照物，卫星是的，以火箭为参照物，卫星是的. 10.小红骑自行车在公路上行驶，当天虽无风，但小红骑在车上觉得刮了西风，以小红为参照物，空气是向运动的，以地面为参照物，小红向行驶. 11. 我们唱的歌“月亮在白莲花般的云朵里穿行”是以为参照物. 12．研究地面上物体的运动时，一般选________做参照物。 13．车里的人看路边的树向后退是以________做参照物。 14．乘客坐在行驶的火车上，以火车为参照物。人是________，以路旁的树木为参照物，人是________的。15．太阳从东方升起，是以________为参照物。月亮在云间穿行，这时我们是以________为参照物。16．以________为参照物，人造地球同步卫星是静止的，以________为参照物，人造地球同步卫星是运动的。

机械运动与参照物习题和答案

初二物理机械运动练习题一、填空题（35分） 1.物理学把 ______________________________叫做机械运动。 2.在研究物体做机械运动时，被选来作为标准的物体叫，同一个物体是运动还是静止的.取决于。这就是运动与静止的相对性. 3.我国古书《套买曜》上记载有：“人在舟中闭牖（门窗）而坐，舟行而人不觉”这是运动的的生动描述，其中“舟行”是以为参照物，“人不觉”是以为参照物. 4.乘客坐在行驶的火车上，以火车为参照物人是的，以路旁的树木为参照物时，人是. 5.平时所说“月亮躲进云里”是以为参照物，说“乌云遮住了月亮”是以为参照物.我国发射的风云二号通讯卫星相对是静止的.相对于是运动的.人们常说地球在公转的同时自转着，“公转”是以为参照物，自转又是以为参照物. 6.空中加油机在空中给歼击机加油的过程中，若以为参照物，他们都是运动的，若以加油机为参照物，歼击机是的. 7.“旭日东升”是以为参照物的，“日落西山”是以为参照物的. 8.坐在甲车里的人，看见路边树木向北运动，他是以为参照物.他看到并排的乙车静止，若以树为参照物，乙车是的. 9.长征三号火箭运载同步卫星升空，此时，以地球为参照物，卫星是的，以火箭为参照物，卫星是的，当卫星脱离火箭绕地球运转时，以地球为参照物，卫星是的，以火箭为参照物，卫星是的. 10.小红骑自行车在公路上行驶，当天虽无风，但小红骑在车上觉得刮了西风，以小红为参照物，空气是向运动的，以地面为参照物，小红向行驶. 11. 我们唱的歌“月亮在白莲花般的云朵里穿行”是以为参照物. 12．研究地面上物体的运动时，一般选________做参照物。 13．车里的人看路边的树向后退是以________做参照物。 14．乘客坐在行驶的火车上，以火车为参照物。人是________，以路旁的树木为参照物，人是________的。15．太阳从东方升起，是以________为参照物。月亮在云间穿行，这时我们是以________为参照物。16．以________为参照物，人造地球同步卫星是静止的，以________为参照物，人造地球同步卫星是运动的。二、判断题(每小题2分，共16分) 1.在研究机械运动时，必须选择地面或地面上静止不动的物体为参照物. （） 2.一个物体是运动的还是静止的，取决于所选取的参照物. （） 3.运动是绝对的，静止是相对的（） 4.自然界中不存在绝对静止的物体. （） 5.我们说小车在平直的公路上做匀速直线运动，这种运动是相对的. （） 6.研究物体做机械运动时，可以任意选择某一物体为参照物，但它一定要是静止的.（） 7.参照物的选取是任意的，但也有一定的原则，必须使研究的问题尽量简单. （） 8.物体甲绕物体己做圆周运动，由于它们之间的距离始终没变，所以甲没有作机械运动. （）三、选择题（每小题2分，共32分） 1.物体的运动和静止都是相对的，都相对于（） A.参照物 B.太阳系 C.太阳 D.地球 2.在飞行的飞机里的人，看到空中的白云迅速地向后退，他所选的参照物是（） A.地面 B.高山 C.飞机 D.白云 3.坐在直升飞机上的驾驶员看见高楼楼顶在向上运动，若以地面为参照物，直升飞机（） A.向下运动 B.已向上运动 C.静止不动 D.水平运动

多项选择题 (4)

请将后面的答案填入题目括号中，以熟悉题目及答案（因为是开卷考试）第一章导论二、多项选择题（1）资产评估行为涉及的经济行为包括（） a、产权转让 b、企业重组 c、资产抵押 d、资产纳税 e、停业整顿（2）资产评估的科学性原则是指（） a、选择适用的价值类型和方法 b、由包括科技专家组成的资产评估队伍 c、制定科学的评估方案 d、以充分科学的事实为依据 e、遵循科学的评估程序（3）资产评估的公正性表现为（） a、资产评估应遵循正确适用的评估原则，依照法定的评估程序，运用科学的评估方法。 b、资产评估主体应当与资产业务及其当事人没有利害关系。 c、资产评估的目标是为了估算出服务于资产业务要求的客观价值。 d、资产评估需要通过对评估基准日的市场实际状况进行模拟。 e评估价值是为资产业务提供的一个参考价值，最终的成交价格取决于资产业务当事人讨价还价的能力。（4）在估价对象已经处于使用状态，运用最佳使用原则的选择前提有（） a、保持利用现状前提 b、转换用途前提 c、投资改造前提 d、新利用前提 e、上述四种情形的某种组合（5）适用于资产评估的假设有（） a、继续使用假设 b、公开市场假设 c、持续经营假设 d、清算假设 e、交易假设（6）确定评估基准日的目的是（） a、确定评估对象的计价时间 b、将动态下的资产固定在某一时点 c、将动态下的资产固定在某一时期 d、确定评估机构的工作日 e、遵循科学的评估程序（1）abcd （2）acde （3）ab （4）abcde （5）abde （6）ab 第二章资产评估的程序与基本方法二、多项选择题（1）资产评估业务约定书的内容包括有（）。 a、评估范围 b、评估目的 c、评估假设 d、评估基准日 e、评估工作日期（2）重置成本的估算方法有（） a、物价指数法 b、功能价值法 c、规模经济效益指数法 d、重置核算法 e、观察法（3）实体性贬值的估算方法有（） a、观察法 b、经济使用年限法 c、使用年限法 d、修复费用法 e、成新率法（4）功能性贬值包括（） a、超额投资性功能性贬值 b、超额运营性功能性贬值 c、复原重置成本的功能性贬值 d、更新重置成本的功能性贬值 e、修复性功能性贬值（5）估算资产净现金流量时所用的资产经营管理成本包括资产（） a、维修费用 b、保险费用 c、折旧费用 d、资金成本 e、管理费用（6）造成资产经济性贬值的主要原因有（）。 a、该项资产技术落后 b、该项资产生产的产品需求减少 c、社会劳动生产率提高 d、自然力作用加剧 e、政府公布淘汰该类资产的时间表（7）复原重置成本与更新重置成本的相同之处在于运用（） a、相同的功能效用 b、相同的建造技术标准 c、资产现时价格

气相色谱中的内标法或外标法(精)

谈谈内标准品（内标物质）传统上在教科书中都会很模糊的告诉学生内标准的选择方式,例如说要选安定性好;与分析物性质要相近;在分析的基质中不能出现等,现在我对这些个" 选择方式"没有太大的兴趣,因为这个大家都知道,那现在就从另一个角度的来看看内标准品的选择还有哪些需要注意的. 1. 只选一个内标准品? 当然, 如果你的分析目标物就只有一个, 在正常的状况下,内标准"应该"也只会有一个才对! 但是如果你的分析是多成份的,那就必须十分小心地看待在一个分析方法内标准品的选择, 如果分析物的在层析图中是平均分布在各处, 那你就必须看看你的检测方法中是否有规定内标物与分析物之间的滞留时间差范围是多少,依此规定来选择内标,但是如果并没有规定,最好也是选择一个以上的内标来使用,因为即使化学性质不会差太多,但在沸点方面却会有满大的差异, 内标与分析物的沸点差异过大,在GC的注射口中就无法把因为discrimibation（分辨）所造成的误差校正回来. 如果你的分析物是性质相差颇大的 (例如说同时含有醇,酸...,那别怀疑一定是要使用一个以上的内标准品,如果多个物种再加上多成份,那就很复杂了. 最低的限度也要依照分析物的沸点高低来使用多个不同的内标准品. 在美国环保署的检验方法USEPA 8270C,是一个检测半挥发性的污染物的规范,前后列了不下一百种的分析物,就使用了六个不同的内标准品作为校正的依据,来照顾到各个不同沸点的分析物!! 滥用药物分析大概是最严谨的了,即使是结构性质极相近的分析物,例如morphine和codeine,amphetamine和methamphetamine,在分析时为求准确,都是以各自的D同位素取代的标准品作为内标. 2.基质中一定不能存在? 这个问题当然是肯定的, 不然定量结果会很不稳定或者是很凄惨. 但是有些时候你根本不知道哪些东西在分析样品的基质中不会存在! 这时候怎么办? 找以往的文献看看别人是用甚么,这是一个方法, 但要注意的是文献不一定就是对的! 使用分析物的氢同位素(D取代物,是最妥当的,但问

色谱定量分析,外标法和内标法如何进行选择

色谱定量分析，外标法和内标法如何进行选择色谱定量分析，外标法和内标法如何进行选择色谱分析的重要作用之一是对样品定量。而色谱法定量的依据是：组分的重量或在载气中的浓度与检测器的响应信号成正比。常见定量分析方法分为面积归一化法、内标法、外标法、标准曲线法等。大家常常容易傻傻分不清楚的莫过于内标法、外标法了。以内标法为例，选一与欲测组分相近但能完全分离的组分做内标物（当然是样品中没有的组分），然后配制欲测组分和内标物的混合标准溶液，进样得相对校正因子。再将内标物加入欲测组分的样品中，进样后测得欲测组分和内标物的定量参数，用内标法公式计算即可。其实，从定义上来区分的话，外标法就是用标准品的峰面积或峰高与其对应的浓度做一条标准曲线，测出样品的峰面积或峰高，在标准曲线上查出其对应的浓度，这是最常用的一种定量方法；内标法是对应外标法说的，内标法是将一定量的纯物质作内标物，加入到准确称量的试样中，根据被测试样和内标物的质量比及其相应的色谱峰面积之比，来计算被测组分的含量。外标法需要用样品和标准品对比，但是有时我们很难保证样品和标准品进的体积是一样的，毕竟要有误差，这时候就用内标法，就是在外标法的基础上,在样品和标准品里在加入一种物质，通过加入物质的峰面积或峰高的变化,就可以看出我们标准品和样品进样体积的差别，但同时会引进加入物质的秤量误差。所以一般用外标法来定量，如果进样体积很难掌握，就用内标法，可以消除进样体积的误差。外标法外标法（标准曲线法、直接比较法）首先用欲测组分的标准样品绘制标准工作曲线。具体作法是：用标准样品配制成不同浓度的标准系列，在与欲测组分相同的色谱条件下，等体积准确量进样，测量各峰的峰面积或峰高，用峰面积或峰高对样品浓度绘制标准工作曲线，此标准工作曲线应是通过原点的直线。若标准工作曲线不通过原点，说明测定方法存在系统误差。标准工作曲线的斜率即为绝对校正因子。当欲测组分含量变化不大，并已知这一组分的大概含量时，也可以不必绘制标准工作曲线，而用单点校正法，即直接比较法定量。单点校正法实际上是利用原点作为标准工作曲线上的另一个点。因此，当方法存在系统误差时（即标准工作曲线不通过原点），单点校正法的误差较大。因此规定，y=ax+b（b的绝对值应不大于100%响应值是y的2%）。标准曲线法的优点：绘制好标准工作曲线后测定工作就很简单了，计算时可直接从标准工作曲线上读出含量，这对大量样品分析十分合适。特别是标准工作曲线绘制后可以使用一段时间，在此段时间内可经常用一个标准样品对标准工作曲线进行单点校正，以确定该标准工作曲线是否还可使用。

(完整版)参照物专项练习(含答案)

运动的描述【课时训练】一、单选题 1.有一首歌的歌词唱道：“月亮在白莲花般的云朵里穿行”，这里选取的参照物是 A. 地面 B. 云朵 C. 人 D. 月亮 2.一人骑自行车由南向北行驶，这时有辆汽车也由南向北从他身旁疾驶而过，若以这辆汽车为参照物，此人 A. 向北运动 B. 向南运动 C. 静止 D. 运动方向无法确定 3.诗句“满眼风光多闪烁，看山恰似走来迎，仔细看山，山不动，是船行.”其中“看山恰似走来迎”和“是船行”所选的参照物依次是 A. 船和山 B. 山和船 C. 山和水 D. 水和山 4.关于机械运动，下列说法正确的是 A. 空气的流动不属于机械运动 B. 运动路线是直的，运动路程是不变的运动才是机械运动 C. 一个物体位置改变就是运动，位置没有改变就是静止 D. 判断一个物体是否运动和怎样运动，离开了参照物就失去了意义 5.一位勇敢的漂流者，坐橡皮船在湍急的河水中顺流而下，对此，下列说法正确的是 A. 以岸边的树为参照物，人是静止的 B. 以船为参照物，河水是流动的 C. 以河水为参照物，人是静止的 D. 以河水为参照物，人是运动的

6.甲看路旁的树木向南运动，乙看甲静止不动，若以树木为参照物，则 A. 甲、乙都向北运动 B. 甲、乙都向南运动 C. 甲向北运动，乙向南运动 D. 甲向南运动，乙向北运动 7.在平直轨道上行驶的一列火车，放在车厢小桌上的茶杯，相对下列哪个物体是运动的 A. 这列火车 B. 坐在车厢椅子上的乘客 C. 关着的车门 D.从旁边走过的列车员二、多选题 8.下列两个物体可认为保持相对静止的是 A. 地球和太阳 B. 一列直线行驶的列车中1 号车厢和5 号车厢 C. 人行走时左脚和右脚 D. 火箭发射离开地面时，火箭和其运载的卫星 9.下列说法中，正确的是 A. 一个物体或物体的一部分，相对于参照物的位置的改变叫运动 B. 乌云遮住了太阳，是以太阳为参照物的 C. 研究某个物体的运动时，可同时选择几个物体做参照物 D. 某同学站着感到没风，当他快跑时，立即感到有风迎面吹来，是以他做参照物的

内标法与外标法

内标法与外标法一、内标法什么叫内标法?怎样选择内标物? 内标法是一种间接或相对的校准方法。在分析测定样品中某组分含量时，加入一种内标物质以校谁和消除出于操作条件的波动而对分析结果产生的影响，以提高分析结果的准确度。内标法在气相色谱定量分析中是一种重要的技术。使用内标法时，在样品中加入一定量的标准物质，它可被色谱拄所分离，又不受试样中其它组分峰的干扰，只要测定内标物和待测组分的峰面积与相对响应值，即可求出待测组分在样品中的百分含量。采用内标法定量时，内标物的选择是一项十分重要的工作。理想地说，内标物应当是一个能得到纯样的己知化合物，这样它能以准确、已知的量加到样品中去，它应当和被分析的样品组分有基本相同或尽可能一致的物理化学性质(如化学结构、极性、挥发度及在溶剂中的溶解度等)、色谱行为和响应特征，最好是被分析物质的一个同系物。当然，在色谱分析条什下，内标物必须能与样品中各组分充分分离。需要指出的是，在少数情况下，分析人员可能比较关心化台物在一个复杂过程中所得到的回收率，此时，他可以使用一种在这种过程中很容易被完全回收的化台物作内标，来测定感兴趣化合物的百分回收率，而不必遵循以上所说的选择原则。在使用内标法定量时，有哪些因素会影响内标和被测组分的峰高或峰面积的比值? 影响内标和被测组分峰高或峰面积比值的因素主要有化学方面的、色谱方面的和仪器方面的三类。由化学方面的原因产生的面积比的变化常常在分析重复样品时出现。化学方面的因素包括： 1、内标物在样品里混合不好； 2、内标物和样品组分之间发生反应， 3、内标物纯度可变等。对于一个比较成熟的方法来说，色谱方面的问题发生的可能性更大一些，色谱上常见的一些问题(如渗漏)对绝对面积的影响比较大，对面积比的影响则要小一些，但如果绝对面积的变化已大到足以使面积比发生显著变化的程度，那么一定有某个重要的色谱问题存在，比如进样量改变太大，样品组分浓度和内标浓度之间有很大的差别，检测器非线性等。进样量应足够小并保持不变，这样才不致于造成检测器和积分装置饱和。如果认为方法比较可靠，而色谱固看来也是正常的话，应着重检查积分装置和设置、斜率和峰宽定位。对积分装置发生怀疑的最有力的证据是：面积比可变，而峰高比保持相对恒定，在制作内标标准曲线时应注意什么? 在用内标法做色话定量分析时，先配制一定重量比的被测组分和内标样品的混合物做色谱分析，测量峰面积，做重量比和面积比的关系曲线，此曲线即为标准曲线。在实际样品分析时所采用的色谱条件应尽可能与制作标准曲线时所用的条件一致，因此，在制作标准曲线时，不仅要注明色谱条件(如固定相、柱温、载气流速等)，还应注明进样体积和内标物浓度。在制作内标标准曲线时，各点并不完全落在直线上，此时应求出面积比和重量比的比值与其平均位的标准偏差，在使用过程中应定期进行单点校正，若所得值与平均值的偏差小于2，曲线仍可使用，若大于2，则应重作曲线，如果曲线在铰短时期内即产生变动，则不宜使用内标法定量。二、外标法

气相色谱中的内标法或外标法

气相色谱中的内标法或外标法谈谈内标准品(内标物质) 传统上在教科书中都会很模糊的告诉学生内标准的选择方式,例如说要选安定性好;与分析物性质要相近;在分析的基质中不能出现等,现在我对这些个" 选择方式"没有太大的兴趣,因为这个大家都知道,那现在就从另一个角度的来看看内标准品的选择还有哪些需要注意的. 1. 只选一个内标准品? 当然, 如果你的分析目标物就只有一个, 在正常的状况下,内标准"应该"也只会有一个才对! 但是如果你的分析是多成份的,那就必须十分小心地看待在一个分析方法内标准品的选择, 如果分析物的在层析图中是平均分布在各处, 那你就必须看看你的检测方法中是否有规定内标物与分析物之间的滞留时间差范围是多少,依此规定来选择内标,但是如果并没有规定,最好也是选择一个以上的内标来使用,因为即使化学性质不会差太多,但在沸点方面却会有满大的差异, 内标与分析物的沸点差异过大,在GC的注射口中就无法把因为discrimibation(分辨)所造成的误差校正回来. 如果你的分析物是性质相差颇大的 (例如说同时含有醇,酸...),那别怀疑一定是要使用一个以上的内标准品,如果多个物种再加上多成份,那就很复杂了. 最低的限度也要依照分析物的沸点高低来使用多个不同的内标准品. 在美国环保署的检验方法USEPA 8270C,是一个检测半挥发性的污染物的规范,前后列了不下一百种的分析物,就使用了六个不同的内标准品作为校正的依据,来照顾到各个不同沸点的分析物!! 滥用药物分析大概是最严谨的了,即使是结构性质极相近的分析物,例如morphine和codeine,amphetamine和methamphetamine,在分析时为求准确,都是以各自的D同位素取代的标准品作为内标.

归一化法,内标法,外标法

归一化法normalization method 一种常用的色谱定量方法。归一化法是把样品中各个组分的峰面积乘以各自的相对校正因子并求和，此和值相当于所有组分的总质量，即所谓“归一”，样品中某组分i的百分含量可用下式计算： pt%= Aifi/(A1f1+A2f2 + ....Anfn )*100 式中f1、f2、fn…为各组分的相对校正因子，A1、A2、…An为各组分的峰面积。如果操作条件稳定，也可以用峰高归一化法定量，此时组分i的百分含量可按下式计算：pt%= hifi/(h1f1+h2f2 + ....hnfn )*100 式中f1、f2、fn、…为各组分在该操作条件下特定的峰高相对校正因子，h1、h2、…hn为各组分的峰高。用归一化法定量时，必须保证样品中所有组分都能流出色谱柱，并在色谱图上显示色谱峰。定量方法色谱中常用的定量方法有： a.校正归一化法当试样中各组分都能流出色谱柱且在检测器上均有响应，各组分的相对校正因子已知时，可用此法定量。组分i在混合物中的百分含量可由下式计算：其中fi可为质量校正因子，也可为摩尔校正因子。若各组分的定量校正因子相近或相同（如同系物中沸点接近的组分），则上式可简化为：该法简称为归一化法。校正归一化法的优点是：简便、准确，当操作条件如进样量、流速变化时，对定量结果影响很小。缺点是：对该法的苛刻要求限制了该法的使用。该法适合于常量物质的定量。 b.内标法所谓内标法是将一定量的纯物质作为内标物，加入到准确称量的试样中，根据被测物和内标物的质量及在色谱图上相应的峰面积比，求出某组分的百分含量。

当只需测定试样中某几各组分时，而且试样中所有组分不能全部出峰时，可用此法。此法适合于微量物质的分析。该法的计算公式如下：其中，f 是被测组分相对于内标物的相对校正因子。 si 该法的优点是：受操作条件的影响较小，定量结果较为准确，使用上不象归一化法那样受到限制。该法的缺点是：每次分析必须准确称量被测物和内标物，不适合于快速分析。内标物的选择十分重要。它应该是试样中不存在的物质；加入的量应接近于被测组分色谱；同时要求内标物的色谱峰位于被测组分色谱附近或几个被测组分色谱峰的中间，并与这些组分完全分离；内标物必须不与样品发生反应等。 c.外标法（定量进样－标准曲线法）所谓外标法就是应用被测组分的纯物质来绘制浓度c对响应值A（h）的标准曲线，然后测试被测样品中被测组分的响应信号（峰面积或峰高），由标准曲线即可查出或通过线型回归计算出其百分含量。该法必须定量进样，即测定标准曲线和测定未知物时，进入色谱中的样品量必须一致。该法的优点是：操作简单，计算方便，缺点是：结果的准确度取决于进样量的重现性和操作条件的稳定性。当被测试样中各组分的浓度变化范围不大时（如工厂的中间控制分析）可不必绘制标准曲线，而用单点校正法。即配制一个与被测组分含量十分接近的浓度为的标准溶液，定量进样，由下式计算被测物的含量。 C S 关于归一化法的一些问题？？归一化法中的校正因子如何得到？？？如果不做，采用面积归一化法的误差大吗？？一般那么多的校正因子不可能一一测出呀？？

内标法及外标法方法、原理、优缺点

An internal standard should be used when performing MS quantitation. An appropriate internal standard will control for extraction, HPLC injection and ionization variability. In a complex matrix it is not uncommon for two different standard levels in SRM integrated plots, at the lower end of the standard curve, to give nearly an identical response. It is only when an internal standard is used that the two points can be differentiated. Some researchers attempt to prepare standard curves and run samples without an internal standard and find moderate success. Often without an internal standard % RSDs of replicates can be as high as 20%. Using an internal standard the % RSDs can be brought down to approximately 2%. We run triplicates at each level of our standard curve. How do I choose an internal standard? The best internal standard is an isotopically labeled version of the molecule you want to quantify. An isotopically labeled internal standard will have a similar extraction recovery, ionization response in ESI mass spectrometry, and a similar chromatographic retention time. If you are performing non-clinical PK quantitation it may be difficult to justify such a standard since a special synthesis of an isotopically labeled standard can be expensive and time consuming. Often if you are working with medicinal chemists they will have a library of compound analogs that can be used as internal standards. These analogs were made in the evolution of the compound to be tested and will be similar to the compound to be quantified and more importantly will be slightly different by parent mass. Try to avoid using de-methylated (-14) or hydroxylated (+16) analogs as internal standards since these are the most common mass shifts observed in naturally occuring metabolites of the parent compound. A common internal standard is a chlorinated version of the parent molecule. A chlorinated version of the parent molecule will commonly have a similar chromatographic retention time which is an important characteristic of an internal standard. We have found that one of the most important characteristics of an internal standard is that it co-elutes with the compound to be quantified.