Soft Lens Measurement

Klaus Ehrmann , in Contact Lens Practice (Third Edition), 2018

Refractive Index – Dispersion

Abbe refractometers are the preferred choice of instrument for measuring the refractive index of hydrogel contact lenses. They are piece of cake to employ and provide the required precision of 0.001. Abbe refractometers mensurate the critical angle of incidence for total internal reflection, which is directly correlated to the refractive index.

Eq. 7.ii n = north ′ × undefined sin α

where due north  =   refractive index of test sample, n′   =   refractive index of reference surface, and α  =   critical bending of incidence upon reference surface.

For the contact lens practitioner, in that location are hand-held instruments on the market (Fig. 7.15), some of which take a scale that converts refractive index into percentage water content for hydrogel materials. Bench-height instruments such as the Abbe Mark III Refractometer (Reichert, NY, USA) and the Standard Abbe Refractometer from Edmund Optics (Barrington, NJ, United states) are generally more accurate. Consideration needs to exist given to the illuminating wavelength as some materials tin can have noticeable dispersion. White light will give an boilerplate over the full visible spectrum. ISO 18369-iv specifies a unmarried wavelength of 546.one nm or 587.6 nm. The CLR12-70 (Alphabetize Instruments Ltd, Peterborough, Britain) is one of the few dedicated instruments for contact lens materials (Fig. seven.16).

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Rigid Lens Measurement

Klaus Ehrmann , in Contact Lens Do (Tertiary Edition), 2018

Refractive Index

The Abbe refractometer is the standard instrument for measuring refractive index of rigid lens materials. This instrument determines the critical incidence angle of a low-cal beam to achieve total internal reflection. The internal reflection is correlated to the refractive index of the optical material. Reliable measurements can be made simply if the contact area betwixt the measurement prism and the test material is at least 3 mm 2. The curved surface of rigid contact lenses is difficult to be measured direct, although Hodur et al. (1992) reported that with a drib of saline as contact fluid, reliable readings tin can exist obtained with the hand-held N3000 refractometer (Atago, Japan). More suitable for refractive index measurements are cloth samples with one apartment polished surface.

ISO 18369-4 (ISO, 2006d) specifies that a wavelength of 546 or 588 nm is to be used. The CLR 12-lxx refractometer (Index Instruments, United kingdom of great britain and northern ireland) is a dedicated musical instrument for rigid and soft contact lens materials that is fully automatic, eliminating operator skills and the subjective variability oftentimes reported with transmission refractometers.

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Urine Specific Gravity

In Clinical Veterinary Advisor: The Horse, 2012

Specimen and Processing Considerations

Lab Artifacts that May Interfere with Readings of Levels of this Substance (and how—Artificially Elevated vs. Depressed)

•

A refractometer should be used for USG measurements. Specific gravity test pads on reagent strips are unreliable in equine urine. When using dipsticks to make up one's mind USG, the post-obit will alter the reading:

○

pH of urine: The reagent strip underestimates USG in alkaline urine.

○

Poly peptide: Urine with a reagent strip reading of i+ to 2+ poly peptide has an overestimated USG.

○

Ketones: Urine with a positive ketone reading (rare in horses) has an overestimated USG.

Sample for Collection (Type of Specimen, Color Tube) and Any Special Specimen Handling Notes

Get-go urine sample voided (or collected via catheterization) earlier fluid therapy or immediately later institution of fluid treatment

Pearls

A wide range of USG tin can be constitute in clinically healthy or sick horses, and concurrent evaluation of hydration status, USG, and serum chemistry values are essential for proper interpretation. A USG without cognition of these aspects is meaningless and collection of urine and serum at the same time is essential.

Writer: LAURA C. CREGAR

EDITOR: CHARLES WIEDMEYER

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Intrinsically Matted Proteins

Timir Baran Sil , ... Kanchan Garai , in Methods in Enzymology, 2018

six.1 FCS Measurements on a Solution of Rhodamine B in Urea

vi.one.1 Equipment and Materials

•

A refractometer (DR-A1, Atago) to measure out the refractive index of the urea solutions

•

The home-built cuvette-FCS

•

Rhodamine B solution (60   due northM) in PBS, pH 7.4

•

Urea (9 Yard) stock solution in PBS, pH 7.4

half dozen.1.2 Process

(i)

Measure the refractive index of the stock solution of urea. Use the measured refractive index to determine the concentration of urea in the solution. We employ the online urea concentration calculator (http://sosnick.uchicago.edu/gdmcl.html). If the concentration is higher than 9 Chiliad, then add appropriate amount of PBS to adjust the concentration to 9 M urea.

(ii)

Prepare a table for the titration of urea in the native buffer. Here nosotros evidence the tabular array that we take used (see Table two).

Table two. Concentration of Urea in the Cuvette With Serial Addition of 9 Grand Urea

Sample Number Total Volume (μL) Volume Withdrawn From Cuvette (μL) Volume of 9 M Urea Added to the Solution (μL) [Urea] (M) Relative Viscosity (cps)
1 2000 0 0 0.00 1.00
ii 2000 100 100 0.45 one.02
3 2000 100 100 0.88 1.04
four 2000 100 100 ane.28 1.05
5 2000 100 100 1.67 ane.07
six 2000 200 200 2.22 1.10
7 2000 200 200 two.73 1.13
8 2000 200 200 iii.20 i.16
nine 2000 200 200 iii.63 1.nineteen
x 2000 300 300 4.17 ane.23
xi 2000 300 300 iv.65 1.27
12 2000 300 300 v.09 ane.31
13 2000 300 300 5.48 1.34
14 2000 400 400 6.01 1.twoscore
fifteen 2000 400 400 6.46 1.44
16 2000 400 400 vi.84 i.49
17 2000 400 400 vii.sixteen 1.53
(iii)

Take a 2   mL solution of threescore   nChiliad rhodamine B in native buffer in the cuvette. Set the temperature of the cuvette holder to the desired temperature. Here we have used 25°C.

(iv)

Use a 0.7 OD ND filter in front of the laser. This gives well-nigh 15   μW excitation power and 6   kHz of CPM per detection aqueduct.

(5)

If this experiment is being performed for the kickoff time, and then we recommend a z-scan, i.e., FCS measurements equally a function of the focal depth (approximately between 0 and 70   μm). Fig. fiveA shows the plot of CPM as a function of the focal depth. As expected the CPM is highest when the focal depth is about 25   μm. Optionally, one may also follow the τ D as a part of focal depth. It may be seen from Fig. fiveB that τ D is the minimum at the position of the highest CPM. Nosotros will then compare this plot with a like experiment at the cease of the titration experiment to examine the stability of the setup and robustness of the measurements.

Fig. 5

Fig. 5. Cuvette-FCS measurements on threescore   nThousand rhodamine B in 0–7 M urea. (A) CPM and (B) τ D vs focal depth in 0 M urea, i.e., at the beginning (squares) and in 7 M urea, i.e., at the end of the experiment (triangles). Clearly, the focal depth remains almost the same during the entire duration of the experiment indicating robustness of the setup. (C) Normalized Grand(τ) curves as a role of τ/η, where η is the viscosity of the solution. (D) CPM increases with the concentration of urea. An opposite tendency would be expected if the ascertainment volume suffers from aberrations in urea. (East) ω remains the same at all concentrations of urea. (D) Improvidence time increases linearly with the viscosity of urea consistent with the Stokes–Einstein relationship.
(vi)

Record 1000(τ) data. Nosotros generally tape x autocorrelation curves at each concentration of urea.

(vii)

Follow Table ii and withdraw the specified volume of the solution from the cuvette. Add the aforementioned volume of 9 Grand urea to keep the full volume same. For example, row 2 in Table 2 shows that in the outset step of the dilution series we withdraw 100 μL of the rhodamine B solution from the cuvette. Save this solution in a centrifuge tube clearly labeled with the sample number and the concentration of urea. This solution may be examined later using the refractometer if any confusion arises about the concentration of urea in the solution. Add equal book (100  μL) of 9 M urea into the cuvette. Mix the solution with a 200   μL pipette gently. Take intendance that the pipette or the tip does non bear on any part of the cuvette or the holder. Let the temperature of the sample equilibrate for ~   2   min. Then perform the FCS measurements as before.

(eight)

Repeat the withdraw/injection cycle as per Table 2 and perform the FCS measurements equally described earlier.

(9)

At the finish of the measurement, i.e., at the highest concentration of urea perform FCS measurements as a office of sample Z-position as described earlier in Pace (v). Record CPM and optionally, τ D every bit a part of the focal depth. Compare this plot with the plot prepared at the offset of the experiment. Data presented in Fig. 5A and B show that the plots of CPM and τ D obtained in the start and at the end of the titration experiments agree quite well. This shows that the cuvette-FCS setup is quite stable.

(x)

Echo the entire experiment at least three times to test reproducibility of the measurements and to calculate the error confined.

6.1.2.1 Notes
1.

Since the viscosity of water is extremely sensitive to the temperature, measurements of τ D may exist severely afflicted if the temperature of the cuvette holder is not maintained properly. Therefore, it is highly important that the temperature of the holder is maintained at a abiding value. In our setup the temperature of the cuvette tin be maintained inside ±   0.5°C of the set temperature. Control of the temperature can be improved further by using PID-based temperature controllers.

ii.

To record CPM as a function of focal depth, it is not necessary to record, salvage, and analyze all the G(τ) information. At each position of the focal depth Thou(0) may be noted from the G(τ) bend by middle estimation just. Mean CR tin can be noted from the time trace of CR by eye estimation. CPM tin can then exist calculated by using the expression CPM   =   CR   × G(0).

iii.

In the process of series addition of urea the concentration of rhodamine B decreases serially. However, τ D and CPM are intrinsic properties; hence these parameters are not affected.

half-dozen.ane.three Assay of the FCS Data

(i)

Summate and plot both the mean and the SD of the G(τ) data from τ  >   10   μs at each concentration of urea as described in Department 5.2.iii.

(ii)

Fit the mean Thou(τ) data using Eq. (iii) (ane-component improvidence model). Make sure that the fitting program uses the SD to optimize the reduced χ 2 as discussed earlier.

(iii)

Record CPM, τ D, and ω at each concentration of urea.

(iv)

A simple style to examine if the G(τ) data have undergone changes in urea is to plot all the normalized G(τ) curves together with the τ normalized by the viscosity (η) of the corresponding solution. For this plot the values of τ demand to be divided past the η of the corresponding urea solution.

half-dozen.1.iv Results

Fig. vC shows the viscosity normalized G(τ) curves obtained in different concentrations of urea. To plot these curves the τ values accept been normalized past the viscosity of the corresponding solution and the G(τ) curves are normalized by the Thou(0). It may exist seen that all the Grand(τ) curves overlap with each other. In Fig. vD and E we have plotted the CPM and the axial ratio (ω) as a function of concentration of urea. Information technology may be seen that the CPM of rhodamine B increases in urea. This is due to the increment of breakthrough yield of the dye in urea (Sahoo et al., 2018). It may be noted here that CPM suffers significantly in instance of optical aberrations in the presence of mismatch of refractive indices (Chattopadhyay et al., 2005; Hell et al., 1993). Therefore, this plot serves every bit a practiced indicator of the quality of the PSF and the robustness of the FCS measurements. Furthermore, it may be seen that ω remains almost abiding within 10   ±   2 over the entire range of the concentration of urea. Once over again it indicates minimal or no aberrations in the PSF. In Fig. 5F, we plot τ D/ τ D,west equally a function of viscosity of urea (η/η due west). The τ D,w and η w are the diffusion time of rhodamine B in water and the viscosity of water, respectively. The values of η at dissimilar concentrations of urea are listed in Table 2 obtained from Kawahara and Tanford (1966). Information technology may be seen here that the τ D/ τ D,w increases linearly with η/η w, consistent with the Stokes–Einstein relationship (Sherman et al., 2008). Taken together results from these experiments bespeak that the cuvette-FCS provides robust measurements of τ D in urea.

6.i.5 Notes

1.

Minimal aberrations due to refractive index mismatch in cuvette-FCS tin be due to the following reason. We are using a low NA objective. Optical aberrations in such cases are shown to be depression (Hell et al., 1993).

2.

While fitting the G(τ) data with Eq. (3) here we have kept ω equally a complimentary parameter. However, sometimes more than consequent values of τ D may exist obtained if the Chiliad(τ) curves at all concentrations of urea are fit using a stock-still value of ω. For this purpose, ω may exist fixed to its estimated boilerplate value (run into Fig. 5E).

3.

The Stokes–Einstein equation is the post-obit:

(12) D = k B T six πη R h

Since τ D is inversely proportional to D (run across Eq. 5), the τ D is expected to exist proportional to η.

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Perinatal Diseases

In Veterinary Medicine (Eleventh Edition), 2017

Serum Full Protein

Measuring total protein concentrations in serum or plasma with a refractometer is a practical, rapid, and inexpensive method to estimate the immunoglobulin concentration by extrapolating information technology from the total protein concentration. Despite the indirect nature of the test, there is a reliable correlation between the refractometer reading and total immunoglobulin concentration measured by RID. In healthy calves a serum total protein of 5.five chiliad/dL or greater is associated with adequate transfer of passive amnesty.

Serum full protein has a skillful predictive value for fate of the newborn, and the facile and practical nature of the test and its predictive ability commend information technology for survey studies in calves and lambs merely not foals. Cutting-points will vary with the environment and the infection pressure to the calves. The sensitivity of the exam is maximal using a cut-bespeak of v.5 g/dL, and the specificity is maximal at a cutting-point of 5.0 g/dL. Because serum full poly peptide concentration measured by refractometry can result in an incidental misclassification of an individual dogie, this test is primarily recommended as a screening tool to assess the colostrum management on a herd level, but not as diagnostic tool for an individual animal. Herd screening could be conducted by testing a minimum of 12 calves on a farm between 24 hours and vii days erstwhile. At least 80% of tested calves should have serum protein concentrations above 5.5 thousand/dL to consider the colostrum management satisfactory at the herd level.

Serum total protein concentration can also be estimated using the same Brix refractometer used for measuring colostral IgG concentration, with an appropriate adjustment cistron. fourteen

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Refractive Alphabetize Measurements

A.F. Rawle , in Encyclopedia of Spectroscopy and Spectrometry (Third Edition), 2017

Food Industry

A variety of relatively low-cost handheld or digital refractometers (a large selection on online sale websites!) discover their niche here catering to food applications such as fats, waxes, and sugar (Brix) — for example, in fermentation, salinity, coffee, and fruit juices. Such instruments exercise not measure concentration straight, although they are often calibrated in such a way. The simple biprism, Abbe-similar, structure provides a dark field and light field in the viewfinder via the disquisitional ray concept (see " Abbe refractometer type " section). A simple solution such equally saccharide in water provides an increase in RI for increasing saccharide concentration, and thus, an instrument can be directly calibrated in % carbohydrate equivalent (1% sugar   =   1°Brix). It is useful to note that the actual solution RI tin can be obtained by using a lookup chart, such as that institute in the CRC Handbook, indicating the RI for various saccharide concentrations, so that these relatively cheap systems practise allow access to the RI. Slightly more complicated systems with temperature control or a full Abbe system allow waxes and fats to be measured in liquid course higher up their melting point. Note that other dissolved solids will also add to the RI increment and care should be taken in interpretation. Sugar in urine and coffee concentration (for the optimum, eighteen–22% dissolved solids) are two other application areas. It is easy to digitize such instruments. These can provide more decimal places and a digital readout simply suffer from the need to accept batteries or an electrical supply. The simplest systems obviously utilise ambient low-cal ( Figs. 1 and ii ).

Figs. 1 and 2

Figs. 1 and 2

Figs. 1 and 2. Handheld refractometer. Alan Rawle: own pictures.

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Collection and Assay of Respiratory Tract Samples

Jennifer L Hodgson , David R Hodgson , in Equine Respiratory Medicine and Surgery, 2007

Biochemical evaluation

Biochemical evaluation is routinely performed only on PF, and non on TAs or BALF. Generally, protein content is measured, though a variety of boosted biochemical variables may be determined when specific pathological conditions are suspected.

•

Total protein content of PF is routinely measured using a refractometer on samples collected into EDTA. Chemic methods (e.g. biuret technique) may exist used, though mostly only in reference laboratories. The full protein concentration of normal PF is <25 1000/fifty (2.5 g/dl) ( DeHeer et al 2002). With small sample volumes (i.eastward. EDTA tubes are less than a quarter total), erroneous results for fluid poly peptide concentrations and cell counts may be obtained. Protein concentrations as adamant past refractive index may be artificially increased (solute consequence of the EDTA), whereas protein concentrations equally determined chemically (due east.g. biuret technique) may be artificially decreased (dilutional outcome).

•

Glucose and lactate concentrations and pH may exist measured to aid in differentiating septic from non-septic pleural exudates. If these analytes are to exist assayed, samples should be collected into tubes containing fluoride–oxalate to prevent cellular metabolism of glucose. Water-soluble molecules of low molecular weight, such as glucose and lactate, readily diffuse from the circulation into PF, resulting in like concentrations in blood and PF in normal horses. Increased anaerobic glycolysis past metabolically active cells (leukocytes or neoplastic cells) or bacterial organisms may subtract glucose concentrations, increase lactate concentrations, and subtract pH. Consequently, decreased PF glucose (<0.iv 1000/l or twoscore mg/dl), increased lactate (greater than a paired blood sample) and decreased pH (<seven.0) are considered by some to exist useful in predicting sepsis, even in horses where PF microbial cultures demonstrate no growth. Pleural fluid glucose concentrations >0.6 thou/l (>lx mg/dl) may be interpreted to suggest an uncomplicated (not-septic) pleural effusion (DeHeer et al 2002).

•

Pleural fluid triglyceride and cholesterol concentrations are useful in distinguishing chylous from pseudochylous effusions. Chylous effusions are characterized by triglyceride concentrations greater than, and cholesterol concentrations less than, paired serum values. Conversely, elevated PF cholesterol and depression triglyceride values are expected in pseudochylous effusions.

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Profiles of Drug Substances, Excipients, and Related Methodology

Nasr Y. Khalil , ... Tanveer A. Wani , in Profiles of Drug Substances, Excipients and Related Methodology, 2019

4.four.ii.one HPLC coupled with refractive index (RI) detector

Duong et al. developed a method in which a differential refractometer detector was used to analyze topiramate sprinkle capsule formulations [23]. The focus of the method was to evaluate the integrity of the polymeric outer coat of the formulation and to indirectly determine the extent of gustatory modality masking. The authors determined the content of topiramate later on a washout procedure of the sprinkle formulation, which consisted of several steps of dipping the formulation into a specific basket of h2o and then analyzing the last solution. The integrity of the coating was directly related to the topiramate content. If the coating of the formulation was satisfactory, very low levels of topiramate would be observed. The method was validated and considered precise, linear over the range 3–sixty   μg/mL, and authentic. The authors applied the method to select the optimal coating conditions. 3 formulations containing different amounts of polymeric coating (9%, eleven% and xiii%) were evaluated, and the integrity of the coating was indirectly determined past evaluating the topiramate content after the blanket process. The method was able to differentiate between different blanket levels, in which the higher level of coating resulted in a lower level of topiramate detected. The authors stated that 11% coating level of the polymeric coat was appropriate to taste masking. The method was besides used to back up the encapsulation of the formulation because the encapsulation process can cause fractures or breakage in the beads; therefore, the method was applied later on coating and so again afterwards the encapsulation. The resulting data were used to optimize the settings on the encapsulation equipment. The authors concluded that the developed method can be used during the conception development in order to optimize the blanket level and also to back up the encapsulation effect of the conception.

In another study using HPLC with RI detection, Biro and co-workers presented 3 different methods to determine the levels of topiramate and its impurities using HPLC with RI detection [24]. In method 1, the authors used isocratic elution to split and quantify topiramate and impurity i (2,3:4,v-bis-O-(i-methylethylidene)-β-d-fructopyranose). This method was not able to separate the most polar impurity 2 (2,3-O-(1-methylethylidene)-β-d-fructopyranose sulfamic acid), which was eluted with the solvent tiptop, nor impurity 4 (N-{[2,3:four,5-bis-O-(1-methylethylidene)-β-fructopyranosyl] oxycarbonyl}-ii,iii:four,five-bis-O-(ane-methylethylidene)-β-d-fructopyranose sulfamic acid), which was co-eluted with impurity three (2,3-O-(one-methylethylidene)-β-d-fructopyranose sulfamic acid) and other unknown impurities. Method 2 combined RI and UV detection with gradient elution to dissever impurities ii, 3 and 4. In this method, impurity two (2,3-O-(i-methylethylidene)-β-d-fructopyranose sulfamic acrid) was separated from the solvent acme and impurity i (2,3:4,5-bis-O-(ane-methylethylidene)-β-d-fructopyranose) in the isocratic part of the run, whereas impurities iii (2,3-O-(one-methylethylidene)-β-d-fructopyranose sulfamic acrid) and 4 (N-{[two,iii:4,5-bis-O-(1-methylethylidene)-β-d-fructopyranosyl] oxycarbonyl}-2,iii:4,5-bis-O-(i-methylethylidene)-β-d-fructopyranose sulfamic acid) were eluted at a higher concentration of the organic modifiers. The authors did not decide the amount of impurity i using method 2, and topiramate was not seen in the chromatogram considering information technology was eluted during the gradient elution part of the run in which RI detection was not possible. Method 3 consisted of anion exchange chromatography coupled simultaneously with UV and inverse RI detection to analyze the inorganic impurities sulfate and sulfamate. The chromatographic conditions were the same as described in the Us Pharmacopeia except for the detector.

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Analysis of Glycans; Polysaccharide Functional Properties

M. Rinaudo , in Comprehensive Glycoscience, 2007

ii.21.3.1.3.ii (ii) Molecular weight determination

Different agaroses were analyzed by size-exclusion chromatography using a differential refractometer and a low-bending laser lite scattering detector; the samples are commencement heated at 90–95  °C (to go the coil conformation for agarose) and injected in the equipment stabilized at 45   °C. The eluent was 0.1   K NaNOthree at the menstruum rate 1ml   min−1. The dn/dc adopted was 0.140. 124

The intrinsic viscosity and the Mark–Houwink parameters were determined in 0.75   K NaSCN at 35   °C; information technology was demonstrated that under these conditions agarose is in the ringlet conformation. 124 The following relation was established:

[5] η = 0.07 Grand 0.72

From this relation, the viscometric average molecular weight can be determined for other agaroses.

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Disorders of Parturition and the Puerperium in the Gilt and Sow

Olli Aarno Peltoniemi , ... Claudio Oliviero , in Veterinarian Reproduction and Obstetrics (Tenth Edition), 2019

Evaluation of Colostrum Quality

Hasan et al. (2016b) have recently proposed the use of Brix refractometer to evaluate the IgG content of sows' colostrum content. Based on the study past Quesnel et al. (2015) and in the review by Hurley (2015), the IgG content in sows peaks inside 2 hours afterward the start of farrowing, with a mean concentration of 64 mg/mL (range: 50–80 mg/mL), whereas it decreases to around x mg/mL at the end of colostrogenesis (24 hours afterward the start of farrowing). By using a Brix measurement on the farm, it might help to identify sows with an impaired IgG concentration and permit improved management of lactating sows and neonate piglets. This procedure should be done during early colostrogenesis (0–three hours afterward start of farrowing), when colostrum is easiest to collect and when superlative levels of IgG occur (Hasan et al. 2016). On the contrary, depression IgG levels (x mg/mL) are non expected to be found during early on colostrogenesis. Taking the lower range of the height IgG level as 50 mg/mL, it should be a adequately reliable betoken to appraise that adequate levels of IgG are nowadays at this stage. Table 17.3 shows a suggestion for the evaluation of the Brix refractometer results. Brix values <20% reflect very low levels of IgG, whereas values >25% reflect good or very good concentrations of IgG. Values between 20% and 24%, divers by the authors as borderline, should not exist considered as being inadequate for the IgG content, especially if the Brix values are in the highest range (23% – 24%). Conversely, levels in the lowest range (20% – 21%) might exist considered more critical. When deadline results are obtained, the authors suggest taking another sample within one to ii hours, to evaluate if the development of the estimated IgG content is stable.

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